WO2011091390A2 - Traitement de maladies liées à la rnase h1 par l'inhibition du produit de transcription naturel antisens de la rnase h1 - Google Patents

Traitement de maladies liées à la rnase h1 par l'inhibition du produit de transcription naturel antisens de la rnase h1 Download PDF

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WO2011091390A2
WO2011091390A2 PCT/US2011/022304 US2011022304W WO2011091390A2 WO 2011091390 A2 WO2011091390 A2 WO 2011091390A2 US 2011022304 W US2011022304 W US 2011022304W WO 2011091390 A2 WO2011091390 A2 WO 2011091390A2
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Prior art keywords
oligonucleotide
rnase
antisense
polynucleotide
nucleic acid
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PCT/US2011/022304
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WO2011091390A3 (fr
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Joseph Collard
Olga Khorkova Sherman
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Opko Curna, Llc
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Priority to NO11735332A priority Critical patent/NO2529015T3/no
Priority to DK11735332.6T priority patent/DK2529015T3/en
Priority to RU2012127702A priority patent/RU2611192C2/ru
Priority to KR1020127018234A priority patent/KR101853510B1/ko
Priority to JP2012550201A priority patent/JP5981850B2/ja
Priority to ES11735332.6T priority patent/ES2671877T3/es
Priority to CA2786568A priority patent/CA2786568A1/fr
Priority to CN2011800062572A priority patent/CN102782135A/zh
Application filed by Opko Curna, Llc filed Critical Opko Curna, Llc
Priority to EP11735332.6A priority patent/EP2529015B1/fr
Priority to US13/575,206 priority patent/US8946182B2/en
Publication of WO2011091390A2 publication Critical patent/WO2011091390A2/fr
Publication of WO2011091390A3 publication Critical patent/WO2011091390A3/fr
Priority to US14/573,326 priority patent/US9745582B2/en
Priority to US15/684,234 priority patent/US10337013B2/en

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    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
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Definitions

  • Embodiments of the invention comprise oligonucleotides modulating expression and/or function of RNASE H I and associated molecules.
  • DNA-RNA and RNA-RNA hybridization are important to many aspects of nucleic acid function including DNA replication, transcription, and translation. Hybridization is also central to a variety of technologies that cither detect a particular nucleic acid or alter its expression. Antisense nucleotides, for example, disrupt gene expression by hybridizing to target RNA, thereby interfering with RNA splicing, transcription, translation, and replication. Antisense DNA has the added feature that DNA-RNA hybrids serve as a substrate for digestion by ribonuclease H, an activity that is present in most cell types.
  • Antisense molecules can be delivered into cells, as is the case for oligodeoxynucleotides (ODNs), or they can be expressed from endogenous genes as RNA molecules.
  • ODNs oligodeoxynucleotides
  • VITRAVENETM for treannent of cytomegalovirus retinitis
  • the invention provides methods for inhibiting the action of a natural antisense transcript by using antisense oligonucleotide(s) targeted to any region of the natural antisense transcript resulting in up-regulation of the corresponding sense gene. It is also contemplated herein that inhibition of the natural antisense transcript can be achieved by siRNA, ribozymes and small molecules, which are considered to be within the scope of the present invention.
  • One embodiment provides a method of modulating function and/or expression of an RNASE H I polynucleotide in patient cells or tissues in vivo or in vitro comprising contacting said cells or tissues with an antisense oligonucleotide 5 to 30 nucleotides in length wherein said oligonucleotide has at least 50% sequence identity to a reverse complement of a polynucleotide comprising 5 to 30 consecutive nucleotides within nucleotides 1 to 437 of SEQ ID NO: 2 or nucleotides 1 to 278 of SEQ ID NO: 3 or nucleotides 1 to 436 of SEQ ID NO: 4 or nucleotides 1 to 1 140 of SEQ ID NO: 5 thereby modulating function and/or expression of the RNASE H I polynucleotide in patient cells or tissues in vivo or in vitro.
  • an oligonucleotide targets a natural antiscnsc sequence of RNASE H I polynucleotides, for example, nucleotides set forth in SEQ ID NOS: 2 to 5, and any variants, alleles, liomologs, mutants, derivatives, fragments and complementary sequences thereto.
  • antisense oligonucleotides are set forth as SEQ ID NOS: 6 to 13.
  • Another embodiment provides a method of modulating function and/or expression of an RNASE H I polynucleotide in patient cells or tissues in vivo or in vitro comprising contacting said cells or tissues with an antiscnsc oligonucleotide 5 to 30 nucleotides in length wherein said oligonucleotide has at least 50% sequence identity to a reverse complement of the an antiscnsc of the RNASE HI polynucleotide; thereby modulating function and or expression of the RNASE H 1 polynucleotide in patient cells or tissues in vivo or in vitro.
  • Another embodiment provides a method of modulating function and/or expression of an RNASE H I polynucleotide in patient cells or tissues in vivo or in vitro comprising contacting said cells or tissues with an antiscnsc oligonucleotide 5 to 30 nucleotides in length wherein said oligonucleotide has at least 50% sequence identity to an antiscnsc oligonucleotide to an RNASE H I antiscnsc polynucleotide; thereby modulating function and/or expression of the RNASE H I polynucleotide in patient cells or tissues in vivo or in vitro.
  • a composition comprises one or more antiscnsc oligonucleotides which bind to sense and/or antisense RNASE H I polynucleotides.
  • the oligonucleotides comprise one or more modified or substituted nucleotides.
  • the oligonucleotides comprise one or more modified bonds.
  • the modified nucleotides comprise modified bases comprising phosphorothioate, methylphosphonate, peptide nucleic acids, 2'-0-methyl, fluoro- or carbon, methylene or other locked nucleic acid (LNA) molecules.
  • the modified nucleotides are locked nucleic acid molecules, including a-L-LNA.
  • the oligonucleotides arc administered to a patient subcutancously, intramuscularly, intravenously or intraperitoneally.
  • the oligonucleotides arc administered in a pharmaceutical composition.
  • a treatment regimen comprises administering the antiscnsc compounds at least once to patient; however, this treatment can be modified to include multiple doses over a period of time.
  • the treatment can be combined with one or more other types of therapies.
  • the oligonucleotides arc encapsulated in a liposome or attached to a carrier molecule (e.g. cholesterol, TAT peptide).
  • a carrier molecule e.g. cholesterol, TAT peptide
  • Figure 1 is a graph of real time PCR results showing the fold change + standard deviation in RNasc H I mRNA after treatment of HepG2 cells with phosphothioatc oligonucleotides introduced using Lipofcctaminc 2000, as compared to control. Bars denoted as CUR-0665, CUR-0666, CUR-0669, CUR-0670, CUR-0667, CUR-0668, CUR- 0671 and CUR-0672 correspond to samples treated with SEQ ID NOS 6 to 13 respectively.
  • RNASEH 1 Homo sapiens ribonuclease H I (RNASEH 1), mRNA (NCBI Accession No.: NM_002936); SEQ ID NO: 2: Natural RNASE HI antisense sequence (Hs.502765); SEQ ID NO: 3: Natural RNASE H I antisense sequence (Hs. 610890); SEQ ID NO: 4: Natural RNASE H I antisense sequence (Hs. 700975); SEQ ID NO: 5: Natural RNASE H I antisense sequence (Hs.606550) SEQ ID NOs: 6 to 13: Antisense oligonucleotides. * indicates phosphothioatc bond.
  • genes, gene names, and gene products disclosed herein are intended to correspond to homologs from any species for which the compositions and methods disclosed herein are applicable.
  • the terms include, but arc not limited to genes and gene products from humans and mice. It is understood that when a gene or gene product from a particular species is disclosed, this disclosure is intended to be exemplary only, and is not to be interpreted as a limitation unless the context in which it appears clearly indicates.
  • die genes disclosed herein which in some embodiments relate to mammalian nucleic acid and amino acid sequences are intended to encompass homologous and/or orthologous genes and gene products from other animals including, but not limited to other mammals, fish, amphibians, reptiles, and birds.
  • the genes or nucleic acid sequences arc human. Definitions
  • the term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within I or more than 1 standard deviation, per the practice in die art. Alternatively, “about” can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to -1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term "about” meaning within an acceptable error range for the particular value should be assumed.
  • mRNA means the presently known mRNA transcript(s) of a targeted gene, and any further transcripts which may be elucidated.
  • antisensc oligonucleotides or “antisensc compound” is meant an RNA or DNA molecule that binds to another RNA or DNA (target RNA, DNA). For example, if it is an RNA oligonucleotide it binds to another RNA target by means of RNA-RNA interactions and alters the activity of the target RNA.
  • An antisensc oligonucleotide can upregulate or downregulate expression and/or function of a particular polynucleotide. The definition is meant to include any foreign RNA or DNA molecule which is useful from a therapeutic, diagnostic, or other viewpoint.
  • Such molecules include, for example, antisensc RNA or DNA molecules, interference RNA (RNAi), micro RNA, decoy RNA molecules, siRNA, enzymatic RNA, therapeutic editing RNA and agonist and antagonist RNA, antisensc oligomcric compounds, antisense oligonucleotides, external guide sequence (EGS) oligonucleotides, alternate splicers, primers, probes, and other oligomcric compounds that hybridize to at least a portion of the target nucleic acid.
  • RNAi interference RNA
  • micro RNA decoy RNA molecules
  • siRNA siRNA
  • enzymatic RNA enzymatic RNA
  • therapeutic editing RNA and agonist and antagonist RNA antisensc oligomcric compounds
  • antisensc oligomcric compounds antisense oligonucleotides
  • EGS external guide sequence oligonucleotides
  • alternate splicers primers, probes,
  • oligonucleotide refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof.
  • oligonucleotide also includes linear or circular oligomers of natural and/or modified monomers or linkages, including dcoxyribonuclcosidcs, ribonuclcosidcs, substituted and alpha-anomeric forms thereof, peptide nucleic acids (PNA), locked nucleic acids (LNA), phosphorothioate, methylphosphonate, and the like.
  • Oligonucleotides are capable of specifically binding to a target polynucleotide by way of a regular pattern of monomer-to-monomer interactions, such as Watson-Crick type of base pairing, Hoogsteen or reverse Hoogsteen types of base pairing, or the like.
  • the oligonucleotide may be "chimeric", that is, composed of different regions.
  • "chimeric" compounds are oligonucleotides, which contain two or more chemical regions, for example, DNA region(s), RNA region(s), PNA region(s) etc. Each chemical region is made up of at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotides compound.
  • These oligonucleotides typically comprise at least one region wherein the oligonucleotide is modified in order to exhibit one or more desired properties.
  • the desired properties of the oligonucleotide include, but are not limited, for example, to increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. Different regions of the oligonucleotide may therefore have different properties.
  • the chimeric oligonucleotides of the present invention can be formed as mixed structures of two or more oligonucleotides, modified oligonucleotides, oligonuclcosides and/or oligonucleotide analogs as described above.
  • the oligonucleotide can be composed of regions that can be linked in "register", that is, when the monomers are linked consecutively, as in native DNA, or linked via spacers.
  • the spacers are intended to constitute a covalent "bridge” between the regions and have in preferred cases a length not exceeding about 100 carbon atoms.
  • the spacers may carry different functionalities, for example, having positive or negative charge, carry special nucleic acid binding properties (intercalators, groove binders, toxins, fluorophors etc.), being lipophilic, inducing special secondary structures like, for example, alanine containing peptides that induce alpha-helices.
  • RNASE H I and "RNase ⁇ are inclusive of all family members, mutants, alleles, fragments, species, coding and noncoding sequences, sense and antisense polynucleotide strands, etc.
  • ribonuclease H I , Rnase H I , rnaseh l , RNASEH I , H I RNA, Ribonuclease H I , Ribonuclease H type 11, RNase H I , RNH I are considered the same in the literature and are used interchangeably in the present application.
  • oligonucleotide specific for or "oligonucleotide which targets” refers to an oligonucleotide having a sequence (i) capable of forming a stable complex with a portion of the targeted gene, or (ii) capable of fomiing a stable duplex with a portion of a mRNA transcript of the targeted gene. Stability of the complexes and duplexes can be determined by theoretical calculations and/or in vitro assays. Exemplary assays for determining stability of hybridization complexes and duplexes are described in the Examples below.
  • target nucleic acid encompasses DNA, RNA (comprising prcmRNA and mRNA) transcribed from such DNA, and also cDNA derived from such RNA, coding, noncoding sequences, sense or antisense polynucleotides.
  • RNA comprising prcmRNA and mRNA
  • cDNA derived from such RNA, coding, noncoding sequences, sense or antisense polynucleotides.
  • antisense The functions of DNA to be interfered include, for example, replication and transcription.
  • RNA to be interfered include all vital functions such as, for example, translocation of the RNA to the site of protein translation, translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and catalytic activity which may be engaged in or facilitated by the RNA.
  • the overall effect of such interference with target nucleic acid function is modulation of the expression of an encoded product or oligonucleotides.
  • RNA interference "RNAi” is mediated by double stranded RNA (dsRNA) molecules that have sequence- specific homology to their "target" nucleic acid sequences.
  • the mediators arc 5-25 nucleotide "small interfering" RNA duplexes (siRNAs).
  • siRNAs are derived from the processing of dsRNA by an RNase enzyme known as Dicer. siRNA duplex products are recruited into a multi-protein siRNA complex termed RISC (RNA Induced Silencing Complex).
  • a RISC is then believed to be guided to a target nucleic acid (suitably mRNA), where the siRNA duplex interacts in a sequence-specific way to mediate cleavage in a catalytic fashion.
  • mRNA target nucleic acid
  • Small interfering RNAs that can be used in accordance with the present invention can be synthesized and used according to procedures that are well known in the art and that will be familiar to the ordinarily skilled artisan.
  • Small interfering RNAs for use in the methods of the present invention suitably comprise between about 1 to about 50 nucleotides (nt).
  • siRNAs can comprise about 5 to about 40 nt, about 5 to about 30 nt, about 10 to about 30 nt, about 1 to about 25 nt, or about 20-25 nucleotides.
  • oligonucleotides are facilitated by using computer programs that automatically align nucleic acid sequences and indicate regions of identity or homology. Such programs are used to compare nucleic acid sequences obtained, for example, by searching databases such as GenBank or by sequencing PCR products. Comparison of nucleic acid sequences from a range of species allows the selection of nucleic acid sequences that display an appropriate degree of identity between species. In the case of genes that have not been sequenced, Southern blots are performed to allow a determination of the degree of identity between genes in target species and other species. By performing Southern blots at varying degrees of stringency, as is well known in the art, it is possible to obtain an approximate measure of identity.
  • enzymatic RNA an RNA molecule with enzymatic activity (Cech, (1 88) J. American. Med. Assoc. 260, 3030-3035).
  • Enzymatic nucleic acids act by first binding to a target RN A. Such binding occurs through the target binding portion of an enzymatic nucleic acid which is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA.
  • the enzymatic nucleic acid first recognizes and then binds a target RNA through base pairing, and once bound to the correct site, acts enzymatiailly to cut the target RNA.
  • decoy RNA is meant an RNA molecule that mimics the natural binding domain for a ligand. The decoy RNA therefore competes with natural binding target for the binding of a specific ligand.
  • TAR HIV trans-activation response
  • the term "monomers” typically indicates monomers linked by phosphodiestcr bonds or analogs thereof to form oligonucleotides ranging in size from a few monomcric units, e.g., from about 3-4, to about several hundreds of monomcric units. Analogs of phosphodiestcr linkages include: phosphorothioate, phosphorodithioate, methylphosphomatcs, phosphoroselcnoate, phosphoramidatc, and the like, as more fully described below. [0038] The term “nucleotide” covers naturally occurring nucleotides as well as nonnaturally occurring nucleotides.
  • nucleotides includes not only the known purine and pyrimidine heterocycles-containing molecules, but also heterocyclic analogues and tautomers thereof.
  • nucleotides are molecules containing adenine, guanine, thymine, cytosine, uracil, purine, xanthine, diaminopurine, 8-oxo- N6-mcthy laden ine, 7-dcazaxanthinc, 7-dcazaguaninc, 4,N4-cthanocytosin, N6,N6- ethano-2,6- diaminopurine, 5-mcthylcytosine, 5-(C3-C6)-alkynylcytosine, 5-fluorouracil, 5-bromouracil, pseudoisocytosine, 2-hydroxy-5-methyl-4-triazolopyridin, isocytosine, isoguanin, inosine and the "non-naturally occuning" nucleotides described in Benner et a/., U.S.
  • nucleotide is intended to cover every and all of these examples as well as analogues and tautomers thereof.
  • Especially interesting nucleotides are those containing adenine, guanine, thymine, cytosine, and uracil, which arc considered as the naturally occurring nucleotides in relation to therapeutic and diagnostic application in humans.
  • Nucleotides include the natural 2'-deoxy and 2'- hydroxyl sugars, e.g., as described in Kornberg and Baker, DNA Replication, 2nd Ed. (Freeman, San Francisco, 1 92) as well as their analogs.
  • nucleotides in reference to nucleotides includes synthetic nucleotides having modified base moieties and/or modified sugar moieties (see e.g., described generally by Scheit, Nucleotide Analogs, John Wiley, New York, 1 80; Freier & Altmann, (1997) N cl. Acid. Res., 25(22), 4429- 4443, Toulme, J.J., (2001) Nature Biotechnology 19: 17-18; Manoharan M., (1999) Biochemicci et Biophysica Ada 1489: 1 17-139; Freier S.
  • Such analogs include synthetic nucleotides designed to enhance binding properties, e.g., duplex or triplex stability, specificity, or the like.
  • hybridization means the pairing of substantially complementary strands of oligomeric compounds.
  • One mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases (nucleotides) of the strands of oligomeric compounds.
  • hydrogen bonding may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding
  • complementary nucleoside or nucleotide bases nucleotides
  • adenine and thymine arc complementary nucleotides which pair through the formation of hydrogen bonds.
  • Hybridization can occur under varying circumstances.
  • An antisense compound is "specifically hybridizable" when binding of the compound to the target nucleic acid interferes with the normal function of the target nucleic acid to cause a modulation of function and/or activity, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target nucleic acid sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and under conditions in which assays are performed in the case of in vitro assays.
  • stringent hybridization conditions refers to conditions under which a compound of the invention will hybridize to its target sequence, but to a minimal number of other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances and in the context of this invention, "stringent conditions" under which oligomeric compounds hybridize to a target sequence are determined by the nature and composition of the oligomeric compounds and the assays in which they are being investigated. In general, stringent hybridization conditions comprise low concentrations ( ⁇ (). ! 5M) of salts with inorganic cations such as Na++ or ++ (i.e., low ionic strength), temperature higher than 20°C - 25° C.
  • the hybridization rate decreases 1.1% for each 1% formamide.
  • An example of a high stringency hybridization condition is 0.1 X sodium chloride-sodium citrate buffer (SSC)/().1 % (w/v) SDS at 60° C. for 30 minutes.
  • Complementary refers to the capacity for precise pairing between two nucleotides on one or two oligomeric strands. For example, if a nucleobase at a certain position of an antisense compound is capable of hydrogen bonding with a nucleobase at a certain position of a target nucleic acid, said target nucleic acid being a DNA, RNA, or oligonucleotide molecule, then the position of hydrogen bonding between the oligonucleotide and the target nucleic acid is considered to be a complementary position.
  • oligomeric compound and the further DNA, RNA, or oligonucleotide molecule are complementary to each other when a sufficient number of complementary positions in each molecule are occupied by nucleotides which can hydrogen bond with each other.
  • “specifically hybridizable” and “complementary” arc terms which are used to indicate a sufficient degree of precise pairing or complementarity over a sufficient number of nucleotides such that stable and specific binding occurs between the oligomeric compound and a target nucleic acid.
  • an oligomeric compound need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable.
  • an oligonucleotide may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure, mismatch or hairpin structure).
  • the oligomeric compounds of the present invention comprise at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 99% sequence complementarity to a target region within the target nucleic acid sequence to which they arc targeted.
  • an antisense compound in which 18 of 20 nucleotides of the antisense compound arc complementary to a target region, and would therefore specifically hybridize would represent 90 percent complementarity.
  • the remaining noncomplcmcntary nucleotides may be clustered or interspersed with complementary nucleotides and need not be contiguous to each other or to complementary nucleotides.
  • an antisense compound which is 18 nucleotides in length having 4 (four) noncomplementary nucleotides which are flanked by two regions of complete complementarity with the target nucleic acid would have 77.8% overall complementarity with the target nucleic acid and would thus fall within the scope of the present invention.
  • Percent complementarity of an antisense compound with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art. Percent homology, sequence identity or complementarity, can be determined by, for example, the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., ( 1 81 ) 2, 482-489).
  • the term "Thermal Melting Point (Tm)” refers to the temperature, under defined ionic strength, pH, and nucleic acid concentration, at which 50% of the oligonucleotides complementary to the target sequence hybridize to the target sequence at equilibrium.
  • stringent conditions will be those in which the salt concentration is at least about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short oligonucleotides (e.g., 10 to 50 nucleotide). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
  • modulation means either an increase (stimulation) or a decrease (inhibition) in the expression of a gene.
  • variants when used in the context of a polynucleotide sequence, may encompass a polynucleotide sequence related to a wild type gene. This definition may also include, for example, "allelic,” “splice,” “species,” or “polymorphic” variants.
  • a splice variant may have significant identity to a reference molecule, but will generally have a greater or lesser number of polynucleotides due to alternate splicing of exons during mRNA processing.
  • the corresponding polypeptide may possess additional functional domains or an absence of domains.
  • Species variants are polynucleotide sequences that vary from one species to another. Of particular utility in the invention arc variants of wild type gene products.
  • Variants may result from at least one mutation in the nucleic acid sequence and may result in altered mRNAs or in polypeptides whose structure or function may or may not be altered. Any given natural or recombinant gene may have none, one, or many allelic fonns. Common mutational changes that give rise to variants are generally ascribed to natural deletions, additions, or substitutions of nucleotides. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.
  • polymorphic variant is a variation in the polynucleotide sequence of a particular gene between individuals of a given species. Polymorphic variants also may encompass "single nucleotide polymorphisms" (SNPs,) or single base mutations in which the polynucleotide sequence varies by one base. The presence of SNPs may be indicative of, for example, a certain population with a propensity for a disease state, that is susceptibility versus resistance.
  • SNPs single nucleotide polymorphisms
  • Derivative polynucleotides include nucleic acids subjected to chemical modification, for example, replacement of hydrogen by an alkyl, acyl, or amino group.
  • Derivatives e.g., derivative oligonucleotides, may comprise non- naturally-occurring portions, such as altered sugar moieties or intcr-sugar linkages. Exemplary among these are phosphorothioatc and other sulfur containing species which are known in the art.
  • Derivative nucleic acids may also contain labels, including radionuclcotidcs, enzymes, fluorescent agents, chcmilumincsccnt agents, chromogenic agents, substrates, cofactors, inhibitors, magnetic particles, and the like.
  • a “derivative" polypeptide or peptide is one that is modified, for example, by glycosylation, pegylation, phosphorylation, sulfation, reduction alkylation, acylation, chemical coupling, or mild formalin treatment.
  • a derivative may also be modified to contain a detectable label, either directly or indirectly, including, but not limited to, a radioisotope, fluorescent, and enzyme label.
  • animal or "patient” is meant to include, for example, humans, sheep, elks, deer, mule deer, minks, mammals, monkeys, horses, cattle, pigs, goats, dogs, cats, rats, mice, birds, chicken, reptiles, fish, insects and arachnids.
  • mammals that are typically under medical care (e.g., humans and domesticated animals). Examples include feline, canine, equine, bovine, and human, as well as just human.
  • Treating covers the treannent of a disease-state in a mammal, and includes: (a) preventing the disease-state from occurring in a mammal, in particular, when such mammal is predisposed to the disease-state but has not yet been diagnosed as having it; (b) inhibiting the disease-state, e.g., arresting it development; and/or (c) relieving the disease-state, e.g., causing regression of the disease state until a desired endpoint is reached. Treating also includes the amelioration of a symptom of a disease (e.g., lessen the pain or discomfort), wherein such amelioration may or may not be directly affecting the disease (e.g., cause, transmission, expression, etc.).
  • a symptom of a disease e.g., lessen the pain or discomfort
  • cancer refers to all types of cancer or neoplasm or malignant tumors found in mammals, including, but not limited to: leukemias, lymphomas, melanomas, carcinomas and sarcomas. The cancer manifests itself as a “tumor” or tissue comprising malignant cells of the cancer.
  • tumors include sarcomas and carcinomas such as, but not limited to: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, cndotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's.
  • sarcomas and carcinomas such as, but not limited to: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, cndotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's.
  • tumor leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadcnocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningi
  • Additional cancers which can be treated by the disclosed composition according to the invention include but not limited to, for example, Hodgkin's Disease, Non-Hodgkin's Lymphoma, multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulincmia, small-cell lung tumors, primary brain tumors, stomach cancer, colon cancer, malignant pancreatic insuianoma, malignant carcinoid, urinary bladder cancer, gastric cancer, prcmalignant skin lesions, testicular cancer, lymphomas, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, cervical cancer, endometrial cancer, adrenal cortical cancer, and prostate cancer.
  • Targets comprise nucleic acid sequences of RNasc HI , including without limitation sense and/or antisense noncoding and/or coding sequences associated with RNASE H 1.
  • RNASE H hydrolyzes RNA in RNA-DNA hybrids.
  • RNasc H activity appears to be ubiquitous in cukaryotes and bacteria. While many mechanisms have been proposed for oligonucleotide mediated destabilization of target RNAs, the primary mechanism by which antisense oligonucleotides are believed to cause a reduction in target RNA levels is through this RNasc H action. Although RNasc H's constitute a family of proteins of varying molecular weight, the nucleolytic activity and substrate requirements appear to be similar for the various isotypes.
  • RNase H's studied to date function as endonucleases exhibiting limited sequence specificity and requiring divalent cations (e.g., Mg.sup.2+, Mn.sup.2+) to produce cleavage products with 5' phosphate and 3' hydroxy! termini in "Nucleases”.
  • divalent cations e.g., Mg.sup.2+, Mn.sup.2+
  • antisense oligonucleotides arc used to prevent or treat diseases or disorders associated with RNASE H I family members.
  • Exemplary RNase H I mediated diseases and disorders which can be treated with cell/tissues regenerated from stem cells obtained using the antisense compounds comprise: leishmaniasis, a hepatic disease or disorder, a disease or disorder associated with mitochondrial dysfunction, cancer, an apoptotic disease, Aicardi-Goutieres syndrome and a retroviral disease or disorder (e.g., AIDS).
  • RNase H I oliginuclcotidcs may further be used in conjunction with other antisense oligonucleotides to increase their activity.
  • modulation of RNASE H I by one or more antisense oligonucleotides is administered to a patient in need thereof, for athletic enhancement and body building.
  • modulation of RNASE H I by one or more antisense oligonucleotides is administered to a patient in need thereof, to prevent or treat any disease or disorder related to R ASE H 1 abnormal expression, function, activity as compared to a normal control.
  • the oligonucleotides arc specific for polynucleotides of RNASE H I , which includes, without limitation noncoding regions.
  • the RNASE H I targets comprise variants of RNASE H I ; mutants of RNASE H I , including SNPs; noncoding sequences of RNASE H I ; alleles, fragments and the like.
  • the oligonucleotide is an antisense RNA molecule.
  • the target nucleic acid molecule is not limited to RNASE H I polynucleotides alone but extends to any of the isoforms. receptors, homologs, non-coding regions and the like of RNASE H I .
  • an oligonucleotide targets a natural antiscnsc sequence (natural antiscnsc to the coding and non-coding regions) of RNASE H I targets, including, without limitation, variants, alleles, homologs, mutants, derivatives, fragments and complementary sequences thereto.
  • tlie oligonucleotide is an antisense RNA or DNA molecule.
  • the oligomcric compounds of the present invention also include variants in which a different base is present at one or more of the nucleotide positions in the compound.
  • the first nucleotide is an adenine
  • variants may be produced which contain thymidine, guanosinc, cytidinc or other natural or unnatural nucleotides at this position. This may be done at any of the positions of the antiscnsc compound. These compounds are then tested using the methods described herein to determine their ability to inhibit expression of a target nucleic acid.
  • homology, sequence identity or complementarity, between the antiscnsc compound and target is from about 50% to about 60%. In some embodiments, homology, sequence identity or complementarity, is from about 60% to about 70%. In some embodiments, homology, sequence identity or complementarity, is from about 70% to about 80%. In some embodiments, homology, sequence identity or complementarity, is from about 80% to about 90%. In some embodiments, homology, sequence identity or complementarity, is about 90%, about 92%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100%.
  • An antisense compound is specifically hybridizabie when binding of the compound to the target nucleic acid interferes with the normal function of the target nucleic acid to cause a loss of activity, and there is a sufficient degree of complementarity to avoid non-specific binding of the antiscnsc compound to non-target nucleic acid sequences under conditions in which specific binding is desired.
  • Such conditions include, i.e., physiological conditions in the case of in vivo assays or therapeutic treatment, and conditions in which assays arc performed in the case of in vitro assays.
  • An antiscnsc compound whether DNA, RNA, chimeric, substituted etc, is specifically hybridizabie when binding of the compound to tlie target DNA or RNA molecule interferes with die normal function of the target DNA or RNA to cause a loss of utility, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and in the case of in vitro assays, under conditions in which the assays arc performed.
  • targeting of RNASE H 1 including without limitation, antisense sequences which are identified and expanded, using for example, PCR, hybridization etc., one or more of the sequences set forth as SEQ ID NOS: 2 to 5, and the like, modulate the expression or function of RNASE H I .
  • expression or function is up-regulated as compared to a control.
  • expression or function is down-regulated as compared to a control.
  • oligonucleotides comprise nucleic acid sequences set forth as SEQ ID NOS: 6 to 13 including antiscnsc sequences which are identified and expanded, using for example, PCR, hybridization etc. These oligonucleotides can comprise one or more modified nucleotides, shorter or longer fragments, modified bonds and the like. Examples of modified bonds or intcmuclcotidc linkages comprise phosphorothioatc, phosphorodithioatc or the like. In an embodiment, the nucleotides comprise a phosphorus derivative.
  • the phosphorus derivative (or modified phosphate group) which may be attached to the sugar or sugar analog moiety in the modified oligonucleotides of the present invention may be a monophosphate, diphosphate, triphosphate, alkylphosphatc, alkancphosphatc, phosphorothioatc and the like.
  • the preparation of the above-noted phosphate analogs, and their incorporation into nucleotides, modified nucleotides and oligonucleotides, per se, is also known and need not be described here.
  • Antisensc oligonucleotides have been employed as therapeutic moieties in the treatment of disease states in animals and man. Antisense oligonucleotides have been safely and effectively administered to humans and numerous clinical trials arc presently underway. It is thus established that oligonucleotides can be useful therapeutic modalities that can be configured to be useful in treatment regimes for treatment of cells, tissues and animals, especially humans.
  • oligomcric antisense compounds bind to target nucleic acid molecules and modulate the expression and/or function of molecules encoded by a target gene.
  • the functions of DNA to be interfered comprise, for example, replication and transcription.
  • the functions of RNA to be interfered comprise all vital functions such as, for example, translocation of the RNA to the site of protein translation, translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and catalytic activity which may be engaged in or facilitated by the RNA.
  • the functions may be up-regulated or inhibited depending on the functions desired.
  • the antisense compounds include, antisense oligomcric compounds, antisense oligonucleotides, external guide sequence (EGS) oligonucleotides, alternate splicers, primers, probes, and other oligomcric compounds thai hybridize to at least a portion of the target nucleic acid.
  • these compounds may be introduced in the form of single-stranded, double-stranded, partially single-stranded, or circular oligomcric compounds.
  • Targeting an antisense compound to a particular nucleic acid molecule can be a multistcp process.
  • the process usually begins with the identification of a target nucleic acid whose function is to be modulated.
  • This target nucleic acid may be, for example, a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a particular disorder or disease state, or a nucleic acid molecule from an infectious agent.
  • the target nucleic acid encodes RNasc H I .
  • the targeting process usually also includes determination of at least one target region, segment, or site within the target nucleic acid for the antisensc interaction to occur such that the desired effect, e.g., modulation of expression, will result.
  • region is defined as a portion of the target nucleic acid having at least one identifiable structure, function, or characteristic.
  • regions of target nucleic acids arc segments.
  • Segments arc defined as smaller or sub-portions of regions within a target nucleic acid.
  • Sites as used in the present invention, are defined as positions within a target nucleic acid.
  • the antisense oligonucleotides bind to die natural antisense sequences of RNase H 1 and modulate the expression and/or function of RNASE HI (SEQ ID NO: 1 ).
  • Examples of antisense sequences include SEQ ID NOS: 2 to 13.
  • the antisense oligonucleotides bind to one or more segments of RNase H I polynucleotides and modulate the expression and/or function of RNASE H I .
  • the segments comprise at least five consecutive nucleotides of the RNASE H 1 sense or antisense polynucleotides.
  • the antisense oligonucleotides arc specific for natural antiscnsc sequences of RNASE H I wherein binding of the oligonucleotides to the natural antiscnsc sequences of RNASE H I modulate expression and/or function of RNASE H I .
  • oligonucleotide compounds comprise sequences set forth as SEQ ID NOS: 6 to 13, antisense sequences which are identified and expanded, using for example, PGR, hybridization etc
  • These oligonucleotides can comprise one or more modified nucleotides, shorter or longer fragments, modified bonds and the like. Examples of modified bonds or intcrmiclcotidc linkages comprise phosphorothioatc, phosphorodithioatc or the like.
  • the nucleotides comprise a phosphorus derivative.
  • the phosphorus derivative (or modified phosphate group) which may be attached to the sugar or sugar analog moiety in the modified oligonucleotides of the present invention may be a monophosphate, diphosphate, triphosphate, alkylphosphatc, alkanephosphatc, phosphorothioatc and the like.
  • the preparation of the above-noted phosphate analogs, and their incorporation into nucleotides, modified nucleotides and oligonucleotides, per sc, is also known and need not be described here.
  • the translation initiation codon is typically 5 -AUG (in transcribed mRNA molecules; 5'-ATG in the corresponding DNA molecule), the translation initiation codon is also referred to as the "AUG codon,” the “start codon” or die “AUG start codon”.
  • a minority of genes has a translation initiation codon having the RNA sequence 5'-GUG, 5'-UUG or 5'-CUG; and 5'-AUA, 5 -ACG and 5'-CUG have been shown to function in vivo.
  • translation initiation codon and “start codon” can encompass many codon sequences, even though the initiator amino acid in each instance is typically methionine (in cukaryotcs) or formylmcthioninc (in prokaryotes).
  • Eukaryotic and prokaryotic genes may have two or more alternative start codons, any one of which may be preferentially utilized for translation initiation in a particular cell type or tissue, or under a particular set of conditions.
  • start codon and “translation initiation codon” refer to the codon or codons that arc used in vivo to initiate translation of an mRNA transcribed from a gene encoding RNase H 1 , regardless of the sequences) of such codons.
  • a translation termination codon (or "stop codon") of a gene may have one of three sequences, i.e., 5'-UAA, 5'-UAG and 5'-UGA (the corresponding DNA sequences are 5 -TAA, 5'- TAG and 5'-TGA, respectively).
  • start codon region and “translation initiation codon region” refer to a portion of such an mR A or gene that encompasses from about 25 to about 50 contiguous nucleotides in cither direction (i.e., 5' or 3') from a translation initiation codon.
  • stop codon region and “translation termination codon region” refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in cither direction (i.e., 5' or 3') from a translation termination codon. Consequently, the "start codon region” (or “translation initiation codon region”) and the “stop codon region” (or 'translation termination codon region”) are all regions that may be targeted effectively with the antisense compounds of the present invention.
  • a targeted region is the intragenic region encompassing the translation initiation or termination codon of the open reading frame (ORF) of a gene.
  • Another target region includes the 5' untranslated region (5'UTR), known in the art to refer to the portion of an mRNA in the 5' direction from the translation initiation codon, and thus including nucleotides between the 5' cap site and the translation initiation codon of an mRN A (or corresponding nucleotides on the gene).
  • Still another target region includes the 3' untranslated region (3'UTR), known in the art to refer to die portion of an mRNA in the 3' direction from the translation termination codon, and thus including nucleotides between the translation termination codon and 3' end of an mRNA (or corresponding nucleotides on the gene).
  • the 5' cap site of an mRNA comprises an N7-methylated guanosinc residue joined to the 5'-most residue of the mRNA via a 5 -5' triphosphate linkage.
  • the 5' cap region of an mRNA is considered to include the 5' cap structure itself as well as the first 50 nucleotides adjacent to the cap site.
  • Another target region for this invention is the 5' cap region.
  • mRNA transcripts Although some cukaryotic mRNA transcripts arc directly translated, many contain one or more regions, known as "introns," which arc excised from a transcript before it is translated. The remaining (and therefore translated) regions are known as “exons" and are spliced together to form a continuous mRNA sequence.
  • targeting splice sites i.e., intron-exon junctions or exon-intron junctions
  • An aberrant fusion junction due to rearrangement or deletion is another embodiment of a target site.
  • mRNA transcripts produced via the process of splicing of two (or more) mRNAs from different gene sources arc known as "fusion transcripts". Introns can be effectively targeted using antisense compounds targeted to, for example, DNA or pre-mRNA.
  • the antisensc oligonucleotides bind to coding and/or non-coding regions of a target polynucleotide and modulate the expression and/or function of the target molecule.
  • the antisensc oligonucleotides bind to natural antisense polynucleotides and modulate the expression and/or function of the target molecule. [0086] In an embodiment, the antisense oligonucleotides bind to sense polynucleotides and modulate the expression and/or function of the target molecule.
  • RNA transcripts can be produced from the same genomic region of DNA. These alternative transcripts are generally known as "variants”. More specifically, “pre-mRNA variants” are transcripts produced from the same genomic DNA that differ from other transcripts produced from the same genomic DNA in either their stan or stop position and contain both intronic and exonic sequence.
  • pre-mRNA variants Upon excision of one or more exon or intron regions, or portions thereof during splicing, pre-mRNA variants produce smaller "mRNA variants". Consequently, mRNA variants are processed pre-mRNA variants and each unique pre-mRNA variant must always produce a unique mRNA variant as a result of splicing. These mRNA variants arc also known as "alternative splice variants”. If no splicing of the pre-mRNA variant occurs then the pre-mRNA variant is identical to the mRNA variant.
  • Variants can be produced through the use of alternative signals to start or stop transcription.
  • Pre-mRNAs and mRNAs can possess more than one start codon or stop codon.
  • Variants that originate from a pre-mRNA or mRNA that use alternative start codons arc known as "alternative start variants" of that pre-mRNA or mRNA.
  • Those transcripts that use an alternative stop codon arc known as "alternative stop variants" of that pre-mRNA or mRNA.
  • One specific type of alternative stop variant is the "polyA variant” in which the multiple transcripts produced result from the alternative selection of one of the "polyA stop signals" by tfic transcription machinery, thereby producing transcripts that terminate at unique polyA sites.
  • the types of variants described herein arc also embodiments of target nucleic acids.
  • the locations on the target nucleic acid to which the antisense compounds hybridize are defined as at least a 5- nuclcotidc long portion of a target region to which an active antisense compound is targeted.
  • Target segments 5- 100 nucleotides in length comprising a stretch of at least five (5) consecutive nucleotides selected from within the illustrative preferred target segments are considered to be suitable for targeting as well.
  • Target segments can include DNA or RNA sequences that comprise at least the 5 consecutive nucleotides from the 5'-tcrminus of one of the illustrative preferred target segments (the remaining nucleotides being a consecutive stretch of the same DNA or RNA beginning immediately upstream of the 5'-terminus of the target segment and continuing until the DNA or RNA contains about 5 to about 100 nucleotides).
  • preferred target segments arc represented by DNA or RNA sequences that comprise at least the 5 consecutive nucleotides from the 3'-tcrminus of one of the illustrative preferred target segments (the remaining nucleotides being a consecutive stretch of the same DNA or RNA beginning immediately downstream of the 3'-tcrminus of the target segment and continuing until the DNA or RNA contains about 5 to about 100 nucleotides).
  • preferred target segments illustrated herein will be able, without undue experimentation, to identify further preferred target segments.
  • antisense compounds are chosen which are sufficiently complementary to the target, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired effect.
  • the oligonucleotides bind to an antisense strand of a particular target.
  • the oligonucleotides arc at least 5 nucleotides in length and can be synthesized so each oligonucleotide targets overlapping sequences such that oligonucleotides are synthesized to cover the entire length of the target polynucleotide.
  • the targets also include coding as well as non coding regions.
  • Targeting an antisense compound to a particular nucleic acid is a multistep process.
  • the process usually begins with the identification of a nucleic acid sequence whose function is to be modulated. This may be, for example, a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a particular disorder or disease state, or a non coding polynucleotide such as for example, non coding RNA (ncRNA).
  • ncRNA non coding RNA
  • RNAs can be classified into ( 1) messenger RNAs (mRNAs), which are translated into proteins, and (2) non- protein-coding RNAs (ncRNAs). ncRNAs comprise microRNAs, antisense transcripts and other Transcriptional Units (TU) containing a high density of stop codons and lacking any extensive "Open Reading Frame". Many ncRNAs appear to start from initiation sites in 3' untranslated regions (3'UTRs) of protein-coding loci. ncRNAs are often rare and at least half of the ncRNAs that have been sequenced by the FANTOM consortium seem not to be polyadenylated.
  • mRNAs messenger RNAs
  • ncRNAs non- protein-coding RNAs
  • TU Transcriptional Units
  • Many ncRNAs appear to start from initiation sites in 3' untranslated regions (3'UTRs) of protein-coding loci. ncRNAs are often rare and at least half of the ncRNAs that have been sequenced by the
  • ncRNAs may regulate gene expression is by base pairing with target transcripts.
  • RNAs that function by base pairing can be grouped into ( ) cis encoded RNAs that arc encoded at the same genetic location, but on the opposite strand to the RNAs they act upon and therefore display perfect complementarity to their target, and (2) trans-encoded RNAs that arc encoded at a chromosomal location distinct from the RNAs they act upon and generally do not exhibit perfect base-pairing potential with their targets.
  • perturbation of an antisense polynucleotide by the antiscnse oligonucleotides described herein can alter die expression of the corresponding sense messenger RNAs.
  • this regulation can either be discordant (antiscnse knockdown results in messenger RNA elevation) or concordant (antisense knockdown results in concomitant messenger RNA reduction).
  • antiscnse oligonucleotides can be targeted to overlapping or non-overlapping parts of the antiscnse transcript resulting in its knockdown or sequestration.
  • Coding as well as non-coding antisense can be targeted in an identical manner and that either category is capable of regulating the corresponding sense transcripts - cither in a concordant or disconcordant manner.
  • the strategies that are employed in identifying new oligonucleotides for use against a target can be based on the knockdown of antisense RNA transcripts by antisense oligonucleotides or any other means of modulating the desired target.
  • Strategy' 2 In the case of concordant regulation, one could concomitantly knock down both antisense and sense transcripts and thereby achieve synergistic reduction of the conventional (sense) gene expression. If, for example, an antisense oligonucleotide is used to achieve knockdown, then this strategy can be used to apply one antisense oligonucleotide targeted to the sense transcript and another antisense oligonucleotide to the corresponding antisense transcript, or a single energetically symmetric antisense oligonucleotide that simultaneously targets overlapping sense and antisense transcripts.
  • antisense compounds include antisense oligonucleotides, ribozymes, external guide sequence (EGS) oligonucleotides, siRNA compounds, single- or double-stranded RNA interference (RNAi) compounds such as siRNA compounds, and other oligomcric compounds which hybridize to at least a portion of the target nucleic acid and modulate its function.
  • GCS external guide sequence
  • siRNA compounds single- or double-stranded RNA interference (RNAi) compounds such as siRNA compounds
  • RNAi RNA interference
  • they may be DNA, RNA, DNA-like, RNA-likc, or mixtures thereof, or may be mimetics of one or more of these.
  • Antisense compounds may be single-stranded, doublcstranded, circular or hairpin oligomcric compounds and may contain structural elements such as internal or terminal bulges, mismatches or loops.
  • Antisense compounds arc routinely prepared linearly but can be joined or otherwise prepared to be circular and/or branched.
  • Antisense compounds can include constructs such as, for example, two strands hybridized to form a wholly or partially double-stranded compound or a single strand with sufficient self- complementarity to allow for hybridization and formation of a fully or partially double-stranded compound.
  • the two strands can be linked internally leaving free 3' or 5' termini or can be linked to form a continuous hairpin structure or loop.
  • the hairpin structure may contain an overhang on cither the 5' or 3' terminus producing an extension of single stranded character.
  • the double stranded compounds optionally can include overhangs on the ends. Further modifications can include conjugate groups attached to one of the termini, selected nucleotide positions, sugar positions or to one of the internucleosidc linkages. Alternatively, the two strands can be linked via a non-nucleic acid moiety or linker group.
  • dsRNA can take the form of a self-complementary hairpin-type molecule that doubles back on itself to form a duplex. Thus, the dsRNAs can be fully or partially double stranded.
  • dsRNA hairpins in transgenic cell lines, however, in some embodiments, the gene expression or function is up regulated.
  • the gene expression or function is up regulated.
  • the two strands (or duplex-forming regions of a single strand) arc complementary RNA strands that base pair in Watson-Crick fashion.
  • nucleic acids including oligonucleotides
  • DNA-like i.e., generally having one or more 2'-dcoxy sugars and, generally, T rather than U bases
  • RNA-like i.e., generally having one or more 2'- hydroxy! or 2'-modificd sugars and, generally U rather than T bases
  • Nucleic acid helices can adopt more than one type of structure, most commonly the A- and B-forms.
  • an antisense compound may contain both A- and B-form regions.
  • tlie desired oligonucleotides or antisense compounds comprise at least one of: antisense RNA, antisense DNA, chimeric antisense oligonucleotides, antisense oligonucleotides comprising modified linkages, interference RNA (RNAi), short interfering RNA (siRNA); a micro, interfering RNA (miRNA); a small, temporal RNA (stRNA); or a short, hairpin RNA (shRNA); small RNA-induced gene activation (RNAa); small activating RNAs (saRNAs), or combinations thereof.
  • RNAi interference RNA
  • siRNA short interfering RNA
  • miRNA micro, interfering RNA
  • shRNA small, temporal RNA
  • shRNA short, hairpin RNA
  • small RNA-induced gene activation RNAa
  • small activating RNAs small activating RNAs (saRNAs), or combinations thereof.
  • dsRNA can also activate gene expression, a mechanism that has been termed "small RNA-induced gene activation" or RNAa.
  • dsRNAs targeting gene promoters induce potent transcriptional activation of associated genes.
  • RNAa was demonstrated in human cells using synthetic dsRNAs, termed “small activating RNAs” (saRNAs). It is currently not known whether RNAa is conserved in other organisms.
  • RNA interference RNA interference
  • siRNA small interfering RNA
  • miRNA microRNA
  • RNAi RNA interference
  • oligonucleotides are shown to increase the expression and/or function of the RNase H I polynucleotides and encoded products thereof.
  • dsRNAs may also act as small activating RNAs (saRNA).
  • saRNA small activating RNAs
  • the "preferred target segments” identified herein may be employed in a screen for additional compounds that modulate die expression of RNase H I polynucleotides.
  • Modules arc those compounds that decrease or increase the expression of a nucleic acid molecule encoding RN ASE ⁇ I and which comprise at least a 5-nucleotide portion that is complementary to a preferred target segment.
  • Tlie screening method comprises the steps of contacting a preferred target segment of a nucleic acid molecule encoding sense or natural antisense polynucleotides of RNASE H I with one or more candidate modulators, and selecting for one or more candidate modulators which decrease or increase the expression of a nucleic acid molecule encoding RNASE H 1 polynucleotides, e.g. SEQ ID NOS: 6 to 13.
  • the candidate modulator or modulators arc capable of modulating (e.g.
  • the modulator may then be employed in further investigative studies of the function of RNASE H I polynucleotides, or for use as a research, diagnostic, or therapeutic agent in accordance with the present invention.
  • Targeting the natural antisense sequence preferably modulates the function of the target gene.
  • the RNASE H I gene e.g. accession number NM 002936.
  • the target is an antisense polynucleotide of the RNASE H I gene.
  • an antisense oligonucleotide targets sense and/or natural antisense sequences of RNASE H I polynucleotides (e.g. accession number NM 002936), variants, alleles, isoforms, homologs, mutants, derivatives, fragments and complementary sequences thereto.
  • the oligonucleotide is an antisense molecule and the targets include coding and noncoding regions of antisense and/or sense RNASE H I polynucleotides.
  • the preferred target segments of the present invention may be also be combined with their respective complementary antisense compounds of the present invention to form stabilized double-stranded (duplexed) oligonucleotides.
  • Such double stranded oligonucleotide moieties have been shown in the art to modulate target expression and regulate translation as well as RNA processing via an antisense mechanism. Moreover, the double-stranded moieties may be subject to chemical modifications. For example, such doublc-strandcd moieties have been shown to inhibit the target by die classical hybridization of antisense strand of the duplex to the target, thereby triggering enzymatic degradation of the target.
  • an antisense oligonucleotide targets RNasc H I polynucleotides (e.g. accession number NM_(K)2936), variants, alleles, isoforms, homologs, mutants, derivatives, fragments and complementary sequences thereto.
  • RNasc H I polynucleotides e.g. accession number NM_(K)2936
  • variants, alleles, isoforms, homologs, mutants, derivatives, fragments and complementary sequences thereto e.g. accession number NM_(K)2936
  • the oligonucleotide is an antisense molecule.
  • the target nucleic acid molecule is not limited to RNASE H 1 alone but extends to any of the isoforms, receptors, homologs and the like of RNASE H 1 molecules.
  • an oligonucleotide targets a natural antisense sequence of RNASE H I polynucleotides, for example, polynucleotides set forth as SEQ ID NOS: 2 to 5, and any variants, alleles, homologs, mutants, derivatives, fragments and complementary sequences thereto.
  • antisense oligonucleotides are set forth as SEQ ID NOS: 6 to 13.
  • the oligonucleotides arc complementary to or bind to nucleic acid sequences of RNASE H I antisense, including without limitation noncoding sense and/or antisense sequences associated with RNASE H I polynucleotides and modulate expression and/or function of RNASE H 1 molecules.
  • the oligonucleotides arc complementary to or bind to nucleic acid sequences of RNASE H I natural antisense, set forth as SEQ ID NOS: 2 to 5 and modulate expression and/or function of RNASE H I molecules.
  • oligonucleotides comprise sequences of at least 5 consecutive nucleotides of SEQ ID NOS: 6 to 13 and modulate expression and/or function of RNASE H I molecules.
  • the polynucleotide targets comprise RNASE H I , including family members thereof, variants of RNASE H I ; mutants of RNASE H 1 , including SNPs; noncoding sequences of RNASE H 1 ; alleles of RNASE H I ; species variants, fragments and the like.
  • the oligonucleotide is an antisense molecule.
  • the oligonucleotide targeting RNASE H I polynucleotides comprise: antisense RNA, interference RNA (RNAi), short interfering RNA (siRNA); micro interfering RNA (miRNA); a small, temporal RNA (stRNA); or a short, hairpin RNA (shRNA); small RNA-induced gene activation (RNAa); or, small activating RNA (saRNA).
  • RNAi interference RNA
  • siRNA short interfering RNA
  • miRNA micro interfering RNA
  • shRNA small, temporal RNA
  • shRNA small RNA-induced gene activation
  • RNAa small activating RNA
  • targeting of RNase H 1 polynucleotides e.g. SEQ ID NOS: 2 to 5 modulate the expression or function of these targets.
  • expression or function is up-regulated as compared to a control.
  • expression or function is down-regulated as compared to a control.
  • antisense compounds comprise sequences set forth as SEQ ID NOS: 6 to 13. These oligonucleotides can comprise one or more modified nucleotides, shorter or longer fragments, modified bonds and the like.
  • SEQ ID NOS: 6 to 13 comprise one or more LNA nucleotides.
  • the modulation of a desired target nucleic acid can be carried out in several ways known in the art. For example, antisense oligonucleotides, siRNA etc.
  • Enzymatic nucleic acid molecules e.g., ribozymes are nucleic acid molecules capable of catalyzing one or more of a variety of reactions, including the ability to repeatedly cleave other separate nucleic acid molecules in a nucleotide base sequence-specific manner. Such enzymatic nucleic acid molecules can be used, for example, to target virtually any RNA transcript.
  • Enzymatic nucleic acid molecules can be designed to cleave specific RNA targets within the background of cellular RNA. Such a cleavage event renders the mRNA non-functional and abrogates protein expression from that RNA. In this manner, synthesis of a protein associated with a disease state can be selectively inhibited.
  • enzymatic nucleic acids with RNA cleaving activity act by first binding to a target RNA. Such binding occurs through the target binding portion of an enzymatic nucleic acid which is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA.
  • the enzymatic nucleic acid first recognizes and then binds a target RNA through complementary base pairing, and once bound to the correct site, acts cnzymatically to cut the target RNA. Strategic cleavage of such a target RNA will destroy its ability to direct synthesis of an encoded protein. After an enzymatic nucleic acid has bound and cleaved its RNA target, it is released from that RNA to search for another target and can repeatedly bind and cleave new targets.
  • RNA-clcaving ribozymcs for the purpose of regulating gene expression.
  • the hammerhead ribozyme functions with a catalytic rate (kcat) of about I min- 1 in the presence of saturating ( 10 mM) concentrations of Mg2+ cofactor.
  • An artificial "RNA ligasc" ribozyme has been shown to catalyze the corresponding self-modification reaction with a rate of about 100 min- 1.
  • Catalytic RNAs designed based on the "hammerhead” motif have been used to cleave specific target sequences by making appropriate base changes in the catalytic RNA to maintain necessary base pairing with the target sequences. This has allowed use of the catalytic RNA to cleave specific target sequences and indicates that catalytic RNAs designed according to the "hammerhead” model may possibly cleave specific substrate RNAs in vivo.
  • RNA interference has become a powerful tool for modulating gene expression in mammals and mammalian cells.
  • This approach requires the delivery of small interfering RNA (siRNA) cither as RNA itself or as DNA, using an expression plasmid or virus and the coding sequence for small hairpin RNAs that are processed to siRNAs.
  • siRNA small interfering RNA
  • This system enables efficient transport of the pre-siRNAs to the cytoplasm where they are active and permit the use of regulated and tissue specific promoters for gene expression.
  • an oligonucleotide or antisensc compound comprises an oligomer or polymer of ribonucleic acid (RNA) and or deoxyribonucleic acid (DNA), or a mimetic, chimera, analog or homolog thereof.
  • RNA ribonucleic acid
  • DNA deoxyribonucleic acid
  • This term includes oligonucleotides composed of naturally occurring nucleotides, sugars and covalcnt intcmuclcosidc (backbone) linkages as well as oligonucleotides having non-naturally occurring portions which function similarly.
  • Such modified or substituted oligonucleotides arc often desired over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for a target nucleic acid and increased stability in the presence of nucleases.
  • the oligonucleotides or "antisense compounds” include antisense oligonucleotides (e.g. RNA, DNA, mimetic, chimera, analog or homolog thereof), ribozymcs, external guide sequence (EGS) oligonucleotides, siRNA compounds, single- or double-stranded RNA interference (RNAi) compounds such as siRNA compounds, saRNA, aRNA. and other oligomeric compounds which hybridize to at least a portion of the target nucleic acid and modulate its function. As such, they may be DNA, RNA, DNA-like, RNA-like, or mixtures thereof, or may be mimetics of one or more of these.
  • antisense oligonucleotides e.g. RNA, DNA, mimetic, chimera, analog or homolog thereof
  • ribozymcs e.g. RNA, DNA, mimetic, chimera, analog or homolog thereof
  • EGS external guide sequence
  • Antisense compounds may be single-stranded, double-stranded, circular or hairpin oligomeric compounds and may contain structural elements such as internal or terminal bulges, mismatches or loops.
  • Antisense compounds are routinely prepared linearly but can be joined or otherwise prepared to be circular and/or branched.
  • Antisense compounds can include constructs such as, for example, two strands hybridized to form a wholly or partially double-stranded compound or a single strand with sufficient self-complementarity to allow for hybridization and formation of a fully or partially double-stranded compound.
  • the two strands can be linked internally leaving free 3' or 5' termini or can be linked to form a continuous hairpin structure or loop.
  • the hairpin structure may contain an overhang on cither the 5' or 3' terminus producing an extension of single stranded character.
  • the double stranded compounds optionally can include overhangs on the ends. Further modifications can include conjugate groups attached to one of the termini, selected nucleotide positions, sugar positions or to one of the intcrnuclcosidc linkages. Alternatively, the two strands can be linked via a non-nucleic acid moiety or linker group.
  • dsRNA can take the form of a self-complementary hairpin-type molecule that doubles back on itself to form a duplex. Thus, the dsRNAs can be fully or partially double stranded.
  • dsRNA hairpins in transgenic cell lines.
  • the two strands are complementary RNA strands that base pair in Watson- Crick fashion.
  • nucleic acids may be described as "DNA-like” (i.e., generally having one or more 2'-dcoxy sugars and, generally, T rather than U bases) or "RNA-like” (i.e., generally having one or more 2'- hydroxy! or 2'-modified sugars and, generally U rather than T bases).
  • Nucleic acid helices can adopt more than one type of structure, most commonly the A- and B-forms.
  • an antiscnsc compound may contain both A- and B-fonti regions.
  • the antisense compounds in accordance with this invention can comprise an antisense portion from about 5 to about 80 nucleotides (i.e. from about 5 to about 80 linked nucleosides) in length. This refers to the length of the antisense strand or portion of the antisense compound.
  • a single-stranded antisense compound of the invention comprises from 5 to about 80 nucleotides
  • a double-stranded antisense compound of the invention (such as a dsRNA, for example) comprises a sense and an antiscnsc strand or portion of 5 to about 80 nucleotides in length.
  • the antisense compounds of die invention have antisense portions of 10 to 50 nucleotides in length.
  • the oligonucleotides are 15 nucleotides in length.
  • the antiscnsc or oligonucleotide compounds of the invention have antisense portions of 12 or 13 to 30 nucleotides in length.
  • antisense compounds having antisense portions of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length, or any range thercwithin.
  • the oligomcric compounds of the present invention also include variants in which a different base is present at one or more of the nucleotide positions in the compound.
  • the first nucleotide is an adenosine
  • variants may be produced which contain thymidine, guanosine or cytidine at this position. This may be done at any of the positions of the antisense or dsRNA compounds. These compounds are then tested using the methods described herein to determine their ability to inhibit expression of a target nucleic acid.
  • homology, sequence identity or complementarity, between the antiscnsc compound and target is from about 40% to about 60%. In some embodiments, homology, sequence identity or complementarity, is from about 60% to about 70%. In some embodiments, homology, sequence identity or complementarity, is from about 70% to about 80%. In some embodiments, homology, sequence identity or complementarity, is from about 80% to about 90%. In some embodiments, homology, sequence identity or complementarity, is about 90%, about 92%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 1 0%.
  • the antisensc oligonucleotides such as for example, nucleic acid molecules set forth in SEQ ID NOS: 2 to 13 comprise one or more substitutions or modifications.
  • the nucleotides arc substituted with locked nucleic acids (LNA).
  • the oligonucleotides target one or more regions of the nucleic acid molecules sense and/or antisensc of coding and or non-coding sequences associated with RNASE H I and the sequences set forth as SEQ ID NOS: 1 to 5.
  • the oligonucleotides are also targeted to overlapping regions of SEQ ID NOS: 1 to 5.
  • Certain preferred oligonucleotides of this invention arc chimeric oligonucleotides.
  • "Chimeric oligonucleotides” or “chimeras,” in the context of this invention, arc oligonucleotides which contain two or more chemically distinct regions, each made up of at least one nucleotide.
  • These oligonucleotides typically contain at least one region of modified nucleotides that confers one or more beneficial properties (such as, for example, increased nuclease resistance, increased uptake into cells, increased binding affinity for the target) and a region that is a substrate for cn2ymes capable of cleaving RNA:DNA or RNA:RNA hybrids.
  • RNase H is a cellular endonuclease which cleaves the R A strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of antisensc modulation of gene expression. Consequently, comparable results can often be obtained with shorter oligonucleotides when chimeric oligonucleotides are used, compared to phosphorothioate deoxyoligonucleotidcs hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.
  • a chimeric oligonucleotide comprises at least one region modified to increase target binding affinity, and, usually, a region that acts as a substrate for RNAse H.
  • Affinity of an oligonucleotide for its target is routinely determined by measuring the Tm of an oligonucleotide/targcl pair, which is the temperature at which the oligonucleotide and target dissociate; dissociation is detected spectrophotometrically. The higher the Tm, the greater is the affinity of the oligonucleotide for the target.
  • Chimeric antisensc compounds of the invention may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosidcs and/or oligonucleotides mimctics as described above. Such; compounds have also been referred to in the art as hybrids or gapmcrs. Representative United States patents that teach the preparation of such hybrid structures comprise, but arc not limited to, US patent nos.
  • the region of the oligonucleotide which is modified comprises at least one nucleotide modified at the 2' position of the sugar, most preferably a 2'-Oalkyl, 2'-0-alkyI-0-alkyl or 2'-fluoro-modified nucleotide.
  • RNA modifications include 2'-fluoro, 2'-amino and 2' O-mcthyl modifications on the ribosc of pyrimidincs, abasic residues or an inverted base at the 3' end of the RNA.
  • RNAse H is a cellular endonuelcase that cleaves the RNA strand of RNA:DNA duplexes; activation of this enzyme therefore results in cleavage of the RNA target, and thus can greatly enhance the efficiency of RNAi inhibition. Cleavage of the RNA target can be routinely demonstrated by gel electrophoresis.
  • the chimeric oligonucleotide is also modified to enhance nuclease resistance.
  • Cells contain a variety of exo- and cndo-nucleascs which can degrade nucleic acids. A number of nucleotide and nucleoside modifications have been shown to make the oligonucleotide into which they arc incorporated more resistant to nuclease digestion than die native oligodeoxynuclcotidc.
  • Nuclease resistance is routinely measured by incubating oligonucleotides with cellular extracts or isolated nuclease solutions and measuring the extent of intact oligonucleotide remaining over time, usually by gel electrophoresis.
  • Oligonucleotides which have been modified to enhance their nuclease resistance survive intact for a longer time than unmodified oligonucleotides.
  • a variety of oligonucleotide modifications have been demonstrated to enhance or confer nuclease resistance.
  • Oligonucleotides which contain at least one phosphorothioate modification are presently more preferred.
  • oligonucleotide modifications which enhance target binding affinity arc also, independently, able to enhance nuclease resistance.
  • oligonucleotides envisioned for this invention include diosc comprising modified backbones, for example, phosphorothioates, phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkyl intcrsugar linkages or short chain hctcroatomic or heterocyclic intcrsugar linkages.
  • oligonucleotides with phosphorothioate backbones and those with heteroatom backbones particularly CH2 --NH— O— CH2, CH, ⁇ N(CH3)--0--CH2 [known as a methylcne(mcthylimino) or MMI backbone], CH2 --0--N (CH3)--CH2, CH2 -N (CH3)-N (CH3)-CH2 and O-N (CH3)-CH2 --CH2 backbones, wherein the native phosphodicstcr backbone is represented as 0--P-0-CH,).
  • the amide backbones disclosed by De Mesmacker et al. ( 1995) Acc. Chem. Res. 28:366-374 are also preferred.
  • oligonucleotides having morpholino backbone structures are also preferred.
  • the phosphodicstcr backbone of the oligonucleotide is replaced with a polyamidc backbone, the nucleotides being bound directly or indirectly to the aza nitrogen atoms of the polyamide backbone.
  • Oligonucleotides may also comprise one or more substituted sugar moieties.
  • Preferred oligonucleotides comprise one of the following at the 2' position: OH, SH, SCH3, F, OCN, OCH3 OCH3, OCH3 0(CH2)n CH3, 0(CH2)n NH2 or 0(CH2)n CH3 where n is from 1 to about 10; C I to CIO lower alkyl, alkoxyalkoxy, substituted lower alkyl, alkaryl or aralkyl; CI; Br; CN; CF3 ; OCF3; 0--, S--, or N-alkyl; 0-, S-, or N-alkcnyl; SOCH3; S02 CH3; ON02; N02; N3; NH2; hctcrocycloalkyl; hctcrocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; an RNA cleaving group; a reporter group; an intercalator, a group for improving the pharmacokinetic
  • a preferred modification includes 2'-methoxyethoxy [2 -0-CH2 CH2 OCH3, also known as 2'-0-(2-mcthoxyethyl)
  • Other preferred modifications include 2'-mcthoxy (2 * -0 ⁇ CH3), 2'- propoxy (2'-OCH2 CH2CH3) and 2'-fluoro (2'-F).
  • Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3' position of the sugar on the 3' terminal nucleotide and the 5' position of 5' terminal nucleotide.
  • Oligonucleotides may also have sugar mimetics such as cyclobutyls in place of the pentofuranosyl group.
  • Oligonucleotides may also include, additionally or alternatively, nucleobase (often referred to in the art simply as “base”) modifications or substitutions.
  • nucleobase often referred to in the art simply as “base”
  • “unmodified” or “natural” nucleotides include adenine (A), guanine (G), thymine (T), cytosine (C) and uracil (U).
  • Modified nucleotides include nucleotides found only infrequently or transiently in natural nucleic acids, e.g., hypoxanthine, 6-methyladenine, 5-Me pyrimidines, particularly 5-mcthylcytosine (also referred to as 5-methyl-2' deoxycytosine and often referred to in the art as 5-Me-C), 5- hydroxymethylcytosine (HMC), glycosyl HMC and gentobiosyl HMC, as well as synthetic nucleotides, e.g., 2- aminoadeninc, 2-(mcthylamino)adcninc, 2-(imidazolylalkyl)adcninc, 2- (aminoalklyamino)adcninc or other heterosubstituted alkyladenines, 2-thiouracil, 2-thiothymine, 5- bromouracil, 5-hydroxymcthyluracil, 8-azaguaninc, 7-
  • a "universal" base known in the art e.g., inosine, may be included.
  • 5-Me-C substitutions have been shown to increase nucleic acid duplex stability by 0.6- 1 ,2°C. and are presently preferred base substitutions.
  • oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates which enhance the activity or cellular uptake of the oligonucleotide.
  • moieties include but arc not limited to lipid moieties such as a cholesterol moiety, a cholesteryl moiety, an aliphatic chain, e.g., dodecandiol or undecyl residues, a polyamine or a polyethylene glycol chain, or Adamantanc acetic acid.
  • Oligonucleotides comprising lipophilic moieties, and methods for preparing such oligonucleotides are known in the art, for example, U.S. Pat Nos. 5, 138,045, 5,218,105 and 5,459,255.
  • oligonucleotides which are chimeric oligonucleotides as hereinbefore defined.
  • the nucleic acid molecule of the present invention is conjugated with another moiety including but not limited to abasic nucleotides, polyethcr, polyamine, polyamides, peptides, carbohydrates, lipid, or polyhydrocarbon compounds.
  • abasic nucleotides polyethcr, polyamine, polyamides, peptides, carbohydrates, lipid, or polyhydrocarbon compounds.
  • these molecules can be linked to one or more of any nucleotides comprising the nucleic acid molecule at several positions on the sugar, base or phosphate group.
  • the oligonucleotides used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including Applied Biosystcms.
  • oligonucleotides Any other means for such synthesis may also be employed; the actual synthesis of the oligonucleotides is well within the talents of one of ordinary skill in the art. It is also well known to use similar techniques to prepare other oligonucleotides such as the phosphorothioatcs and alkylated derivatives.
  • CPG controllcd-porc glass
  • LNA monomers to enhance the potency, specificity and duration of action and broaden the routes of administration of oligonucleotides comprised of current chemistries such as MOE, ANA, FAN A, PS etc. This can be achieved by substituting some of the monomers in the current oligonucleotides by LNA monomers.
  • the LNA modified oligonucleotide may have a size similar to the parent compound or may be larger or preferably smaller.
  • LNA-modified oligonucleotides contain less than about 70%, more preferably less than about 60%, most preferably less than about 50% LNA monomers and that their sizes are between about 5 and 25 nucleotides, more preferably between about 12 and 20 nucleotides.
  • Preferred modified oligonucleotide backbones comprise, but not limited to, phosphorothioatcs, chiral phosphorothioatcs, phosphorodithioatcs, phosphotriestcrs, amiiioalkylphosphotriestcrs, methyl and other alkyl phosphonatcs comprising 3'alkylenc phosphonates and chiral phosphonates, phosphinatcs, phosphoramidatcs comprising 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphatcs having normal 3 -5' linkages, 2'- 5' linked analogs of these, and those having inverted polarity wherein the ad jacent pairs of nucleoside units are linked 3'-5' to 5'- 3' or 2'-5'
  • Preferred modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl intemucleoside linkages, mixed hetcroatom and alkyl or cycloalkyl intemucleoside linkages, or one or more short chain hctcroatomic or heterocyclic intemucleoside linkages.
  • These comprise those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene fomiacetyl and thioformacetyl backbones; alkene containing backbones; sulfamatc backbones; mcthylcneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts.
  • siloxane backbones siloxane backbones
  • sulfide, sulfoxide and sulfone backbones formacetyl and thioformacetyl backbones
  • methylene fomiacetyl and thioformacetyl backbones al
  • both the sugar and the intemucleosidc linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups.
  • the base units arc maintained for hybridization with an appropriate nucleic acid target compound.
  • One such oligomcric compound an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA).
  • PNA peptide nucleic acid
  • the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminocthylglycinc backbone.
  • nucleobascs are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.
  • Representative United States patents that teach the preparation of PNA compounds comprise, but are not limited to, US patent nos. 5,539,082; 5,714,331 ; and 5,719,262, each of which is herein incorporated by reference . Further teaching of PNA compounds can be found in Nielsen, et al. ( 1 1 ) Science 254, 1497-1500.
  • the oligonucleotides with phosphorothioate backbones and oligonuclcosidcs with hcteroatom backbones and in particular- CH2-NH-0-CH2-,-CH2-N (CH3)-0-CH2-known as a methylene (methylimino) or I backbone,- CH2-0- (CH3)-CH2-,-CH2N(CH3)-N(CH3) CH2-and-0-N(CH3)- CH2-CH2- wherein the native phosphodiestcr backbone is represented as-0-P-0-CH2- of the above referenced US patent no. 5,489,677, and the amide backbones of the above referenced US patent no. 5,602,240.
  • Modified oligonucleotides may also contain one or more substituted sugar moieties.
  • Preferred oligonucleotides comprise one of the following at the 2' position: OH; F; 0-, S-, or N-alkyl; 0-, S-, or N-alkenyl; 0-, S- or N-alkynyl; or O alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C to CO alkyl or C2 to CO alkenyl and alkynyl.
  • n and in can be from 1 to about 10.
  • oligonucleotides comprise one of the following at the 2' position: C to CO, (lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, CI, Br, CN, CF3, OCF3, SOCH3, S02CH3, ON02, N02, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the phannacodynamic properties of an oligonucleotide, and other substituents having similar properties.
  • a preferred modification comprises 2'-methoxyethoxy (2'-0-CH2CH20CH3, also known as 2'-0-(2- methoxyethyl) or 2'-MOE) i.e., an alkoxyalkoxy group.
  • a further preferred modification comprises 2'-dimcthylaminooxycthoxy, i.e.
  • a 0(CH2)20N(CH3)2 group also known as 2 -DMAOE, as described in examples herein below
  • 2'- dimcthylaminocthoxycthoxy also known in the art as 2'-0-dimcthylaminocthoxycthyl or 2'- DMAEOE
  • 2'-0-CH2-0-CH2-N CH2
  • Oligonucleotides may also comprise nucleobase (often referred to in the art simply as “base”) modifications or substitutions.
  • base nucleobase
  • "unmodified” or “natural” nucleotides comprise the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
  • Modified nucleotides comprise other synthetic and natural nucleotides such as 5-mcthylcytosine (5-mc-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2- aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5- propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pscudo-uracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5- trifluoromcthyl
  • nucleotides comprise those disclosed in United States Patent No. 3,687,808, those disclosed in The Concise Encyclopedia of Polymer Science And . Engineering', pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., 'Angewandle Chemie, International Edition', 1 91 , 30, page 613, and those disclosed by Sanghvi, Y.S., Chapter 15, 'Antisense Research and Applications', pages 289-302, Crookc, ST. and Lebleu, B. ca., CRC Press, 1993.
  • nucleotides are particularly useful for increasing the binding affinity of the oiigomcric compounds of the invention.
  • These comprise 5-substituted pyrimidines, 6- azapyrimidines and N-2, N-6 and 0-6 substituted purines, comprising 2-aminopropyladenine, 5- propynyluracil and 5-propynylcytosinc.
  • 5- methylcytosine substitutions have been shown to increase nucleic acid duplex stability by () .6-1.2°C (Sanghvi, Y.S., Crookc, ST. and Lebleu, B., cds, 'Antisense Research and Applications', CRC Press, Boca Raton, 1993, pp.
  • Another modification of the oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates, which enhance the activity, cellular distribution, or cellular uptake of the oligonucleotide.
  • Such moieties comprise but arc not limited to, lipid moieties such as a cholesterol moicty, cholic acid, a thioethcr, e.g., hexyl-S-tritylthiol, a thiocholcstcrol, an aliphatic chain, e.g., dodecandiol or undccyl residues, a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium l ,2-di-0-hexadecyl-rac-glyccro-3-H-phosphonate, a polyamine or a polyethylene glycol chain, or Adamantanc acetic acid, a palmityl moicty, or an octadccylamine or hexylamino-carbonyl-t oxycholesterol moiety.
  • lipid moieties such as a cholesterol moi
  • Drug discovery The compounds of the present invention can also be applied in the areas of drug discovery and target validation.
  • the present invention comprehends the use of the compounds and preferred target segments identified herein in drug discovery efforts to elucidate relationships that exist between RNase H I polynucleotides and a disease state, phenotype, or condition.
  • RNASE H I polynucleotides comprising contacting a sample, tissue, cell, or organism with the compounds of the present invention, measuring the nucleic acid or protein level of RNASE H I polynucleotides and/or a related phenotypic or chemical endpoint at some time after treatment, and optionally comparing the measured value to a non-treated sample or sample treated with a further compound of the invention.
  • These methods can also be performed in parallel or in combination with other experiments to determine the function of unknown genes for the process of target validation or to determine the validity of a particular gene product as a target for treatment or prevention of a particular disease, condition, or phenotype.
  • Transfer of an exogenous nucleic acid into a host cell or organism can be assessed by directly detecting the presence of the nucleic acid in the cell or organism. Such detection can be achieved by several methods well known in the art. For example, the presence of the exogenous nucleic acid can be detected by Southern blot or by a polymerase chain reaction (PGR) technique using primers that specifically amplify nucleotide sequences associated with the nucleic acid. Expression of the exogenous nucleic acids can also be measured using conventional metliods including gene expression analysis. For instance, mRNA produced from an exogenous nucleic acid can be detected and quantified using a Northern blot and reverse transcription PCR (RT-PCR).
  • RT-PCR Northern blot and reverse transcription PCR
  • RNA from the exogenous nucleic acid can also be detected by measuring an enzymatic activity or a reporter protein activity.
  • antisensc modulatory activity can be measured indirectly as a decrease or increase in target nucleic acid expression as an indication that the exogenous nucleic acid is producing the effector RNA.
  • primers can be designed and used to amplify coding regions of the target genes. Initially, the most highly expressed coding region from each gene can be used to build a model control gene, although any coding or non coding region can be used. Each control gene is assembled by inserting each coding region between a reporter coding region and its poly(A) signal.
  • plasmids would produce an mRNA with a reporter gene in the upstream portion of the gene and a potential RNAi target in the 3' non-coding region.
  • the effectiveness of individual antisensc oligonucleotides would be assayed by modulation of the reporter gene.
  • Reporter genes useful in the methods of die present invention include acetohydroxyacid synthase (AHAS), alkaline phosphatase (AP), beta galactosidasc (LacZ), beta glucoronidasc (GUS), chloramphenicol acctyltransferase (CAT), green fluorescent protein (GFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), horseradish peroxidase (HRP), luciferasc (Luc), nopalinc synthase (NOS), octopinc synthase (OCS), and derivatives thereof.
  • AHAS acetohydroxyacid synthase
  • AP alkaline phosphatase
  • LacZ beta galactosidasc
  • GUS beta glucoronidasc
  • CAT chloramphenicol acctyltransferase
  • GFP green fluorescent protein
  • RFP red fluorescent protein
  • YFP yellow fluorescent protein
  • Multiple selectable markers are available that confer resistance to ampicillin, bleomycin, chloramphenicol, gentamycin, hygromycin, kanamycin, lincomycin, methotrexate, phosphinothricin, puromycin, and tetracycline.
  • Methods to determine modulation of a reporter gene include, but are not limited to, fluorometric methods (e.g. fluorescence spectroscopy, Fluorescence Activated Cell Sorting (FACS), fluorescence microscopy), antibiotic resistance determination.
  • RNASE H I protein and mRNA expression can be assayed using methods known to those of skill in the art and described elsewhere herein.
  • immunoassays such as the EL1SA can be used to measure protein levels.
  • RNASE H I ELISA assay kits are available commercially, e.g., from R&D Systems (Minneapolis, MN).
  • RNASE H I expression (e.g., mRNA or protein) in a sample (e.g., cells or tissues in vivo or in vitro) treated using an antisensc oligonucleotide of the invention is evaluated by comparison with RNASE H I expression in a control sample.
  • expression of the protein or nucleic acid can be compared using methods known to those of skill in the art with that in a mock-treated or untreated sample.
  • comparison with a sample treated with a control antisensc oligonucleotide e.g., one having an altered or different sequence
  • a difference in the expression of the R ASE H I protein or nucleic acid in a treated vs. an untreated sample can be compared with the difference in expression of a different nucleic acid (including any standard deemed appropriate by the researcher, e.g., a housekeeping gene) in a treated sample vs. an untreated sample.
  • RNASE H I mRNA or protein, in a sample treated with an antiscnsc oligonucleotide of the present invention is increased or decreased by about 1.25-fold to about 10-fold or more relative to an untreated sample or a sample treated with a control nucleic acid.
  • the level of RNASE H I mRNA or protein is increased or decreased by at least about 1.25-fold, at least about 1.3-fold, at least about 1.4-fold, at least about 1.5-fold, at least about 1.6-fold, at least about 1.7-fold, at least about 1.8-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5- fold, at least about 5-fold, at least about 5.5-fold, at least about 6-fold, at least about 6.5-fold, at least about 7-fold, at least about 7.5-fold, at least about 8-fold, at least about 8.5-fold, at least about 9-fold, at least about 9.5-fold, or at least about 10-fold or more.
  • the compounds of the present invention can be utilized for diagnostics, therapeutics, and prophylaxis, and as research reagents and components of kits. Furthermore, antiscnsc oligonucleotides, which are able to inhibit gene expression with exquisite specificity, arc often used by those of ordinary skill to elucidate the function of particular genes or to distinguish between functions of various members of a biological pathway.
  • the compounds of the present invention for use in kits and diagnostics and in various biological systems, the compounds of the present invention, either alone or in combination with other compounds or therapeutics, arc useful as tools in differential and/or combinatorial analyses to elucidate expression patterns of a portion or the entire complement of genes expressed within cells and tissues.
  • biological system or “system” is defined as any organism, cell, cell culture or tissue that expresses, or is made competent to express products of the RNasc H I genes. These include, but arc not limited to, humans, transgenic animals, cells, cell cultures, tissues, xenografts, transplants and combinations thereof.
  • expression patterns within cells or tissues treated with one or more antiscnsc compounds are compared to control cells or tissues not treated with antisensc compounds and the patterns produced arc analyzed for differential levels of gene expression as they pertain, for example, to disease association, signaling pathway, cellular localization, expression level, size, structure or function of the genes examined. These analyses can be pcrfonned on stimulated or unstimulated cells and in the presence or absence of other compounds that affect expression patterns.
  • Examples of methods of gene expression analysis known in the art include DNA arrays or microarrays, SAGE (serial analysis of gene expression), READS (restriction enzyme amplification of digested cDNAs), TOGA (total gene expression analysis), protein arrays and proteomics, expressed sequence tag (EST) sequencing, subtractive RNA fingerprinting (SuRF), subtractive cloning, differential display (DD), comparative genomic hybridization, FISH (fluorescent in situ hybridization) techniques and mass spectrometry methods.
  • RNASE H I modulators are effective primers or probes under conditions favoring gene amplification or detection, respectively. These primers and probes are useful in methods requiring the specific detection of nucleic acid molecules encoding RNASE H I and in the amplification of said nucleic acid molecules for detection or for use in further studies of RNASE H I .
  • Hybridization of the antisense oligonucleotides, particularly the primers and probes, of the invention with a nucleic acid encoding RNASE H I can be detected by means known in the art. Such means may include conjugation of an enzyme to the oligonucleotide, radiolabeling of the oligonucleotide, or any other suitable detection means. Kits using such detection means for detecting the level of R ASE H I in a sample may also be prepared.
  • antisense compounds have been employed as therapeutic moieties in the treatment of disease states in animals, including humans.
  • Antisense oligonucleotide drugs have been safely and effectively administered to humans and numerous clinical trials arc presently underway. It is thus established that antisense compounds can be useful therapeutic modalities that can be configured to be useful in treatment regimes for the treatment of cells, tissues and animals, especially humans.
  • an animal preferably a human, suspected of having a disease or disorder which can be treated by modulating the expression of RNASE H I polynucleotides is treated by administering antisense compounds in accordance with this invention.
  • the methods comprise the step of administering to the animal in need of treatment, a therapeutically effective amount of RNASE H I modulator.
  • the RNASE HI modulators of the present invention effectively modulate the activity of die RNASE H I or modulate the expression of the RNASE H I protein.
  • the activity or expression of RNASE H I in an animal is inhibited by about 10% as compared to a control.
  • the activity or expression of RNASE H 1 in an animal is inhibited by about 30%. More preferably, the activity or expression of RNASE H I in an animal is inhibited by 50% or more.
  • the oligomeric compounds modulate expression of RNase H 1 mRN A by at least 10%, by at least 50%, by at least 25%, by at least 30%, by at least 40%, by at least 50%, by at least 60%, by at least 70%, by at least 75%, by at least 80%, by at least 85%, by at least 90%, by at least 95%, by at least 98%, by at least 99%, or by 100% as compared to a control.
  • the activity or expression of RNasc HI and/or in an animal is increased by about 10% as compared to a control.
  • the activity or expression of RNASE H 1 in an animal is increased by about 30%. More preferably, the activity or expression of RNASE H 1 in an animal is increased by 50% or more.
  • the oligomeric compounds modulate expression of RNASE H I mRNA by at least 1 %, by at least 50%, by at least 25%, by at least 30%, by at least 40%, by at least 50%, by at least 60%, by at least 70%, by at least 75%, by at least 80%, by at least 85%, by at least 90%, by at least 95%, by at least 98%, by at least 99%, or by 100% as compared to a control.
  • the reduction of the expression of RNasc H 1 may be measured in serum, blood, adipose tissue, liver or any other body fluid, tissue or organ of the animal.
  • the cells contained within said fluids, tissues or organs being analyzed contain a nucleic acid molecule encoding RNASE H I peptides and/or the RNASE H 1 protein itself.
  • the compounds of the invention can be utilized in pharmaceutical compositions by adding an effective amount of a compound to a suitable pharmaceutically acceptable diluent or carrier. Use of the compounds and methods of the invention may also be useful prophylactically.
  • Another modification of the oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates that enhance the activity, cellular distribution or cellular uptake of the oligonucleotide.
  • moieties or conjugates can include conjugate groups covalcntly bound to functional groups such as primary or secondary hydroxyl groups.
  • Conjugate groups of the invention include intercalators, reporter molecules, polyamines, polyamidcs, polyethylene glycols, polyethers, groups that enhance the phannacodynamic properties of oligomers, and groups that enhance the phannacokinetic properties of oligomers.
  • Typicalconjugate groups include cholcsterols, lipids, phospholipids, biotin, phenazinc, folate, phenanthridine, anthraquinonc, acridinc, fluoresceins, rhodamincs, coumarins, and dyes.
  • Groups that enhance the phannacodynamic properties include groups that improve uptake, enhance resistance to degradation, and/or strengthen sequence- specific hybridization with the target nucleic acid.
  • Conjugate moieties include, but are not limited to, lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexyl-5- tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., di-hexadccyl-rac-glyccrol or triethylammonium l ,2-di -hcxadecyl-rac-glycero-3-Hphosphonate, a polyaminc or a polyethylene glycol chain, or Adamantane acetic acid, a palmityl moiety, or an octadecylamine or
  • Oligonucleotides of the invention may also be conjugated to active drug substances, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofcn, dansylsarcosinc, 2,3,5-triiodobcnzoic acid, flufcnamic acid, folinic acid, a bcnzothiadiazidc, chlorothiazide, a diazepinc, indomcthicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic.
  • active drug substances for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranopro
  • the compounds of the invention may also be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as forexample, liposomes, receptor-targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption.
  • Representative United States patents that teach the preparation of such uptake, distribution and/or absorption-assisting formulations include, but are not limited to, U.S. Pat. Nos. 5, 108,921 ; 5,354,844; 5,4 16,016; 5.459.127; 5,521 ,2 1 ;
  • antisense oligonucleotides do not need to be administered in the context of a vector in order to modulate a target expression and/or funetion
  • embodiments of the invention relates to expression vector constructs for the expression of antisense oligonucleotides, comprising promoters, hybrid promoter gene sequences and possess a strong constitutive promoter activity, or a promoter activity which can be induced in the desired case.
  • invention practice involves administering at least one of the foregoing antisense oligonucleotides with a suitable nucleic acid delivery system.
  • a suitable nucleic acid delivery system includes a non-viral vector operably linked to the polynucleotide.
  • nonviral vectors include the oligonucleotide alone (e.g. any one or more of SEQ ID NOS: 6 to 13) or in combination with a suitable protein, polysaccharide or lipid formulation.
  • suitable nucleic acid deliver ' systems include viral vector, typically sequence from at least one of an adenovirus, adenovirus-associated virus (AAV), helper-dependent adenovirus, retrovirus, or hcmagglutinatin virus of Japan-liposome (HVJ) complex.
  • the viral vector comprises a strong eukaryotic promoter operably linked to the polynucleotide e.g., a cytomegalovirus (CM V) promoter.
  • CM V cytomegalovirus
  • retroviral vectors include Moloney murine leukemia viruses and HIV-based viruses.
  • One preferred HlV-bascd viral vector comprises at least two vectors wherein the gag and pol genes are from an HIV genome and the env gene is from another virus.
  • DNA viral vectors are preferred. These vectors include pox vectors such as orthopox or avipox vectors, herpesvirus vectors such as a herpes simplex I virus (HSV) vector, Adenovirus Vectors and Adeno-associated Vims Vectors.
  • pox vectors such as orthopox or avipox vectors
  • herpesvirus vectors such as a herpes simplex I virus (HSV) vector
  • Adenovirus Vectors and Adeno-associated Vims Vectors.
  • the antisensc compounds of the invention encompass any phannaccutically acceptable salts, esters, or salts of such esters, or any other compound which, upon administration to an animal, including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof.
  • pharmaceutically acceptable salts refers to physiologically and pharmaceutically acceptable salts of the compounds of the invention: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto.
  • pharmaceutically acceptable salts for oligonucleotides, preferred examples of pharmaceutically acceptable salts and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein by reference.
  • the present invention also includes pharmaceutical compositions and formulations that include the antisensc compounds of the invention.
  • the pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer, intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration.
  • administration can be made by, e.g., injection or infusion into the cerebrospinal fluid.
  • Administration of antisensc RNA into cerebrospinal fluid is described, e.g., in U.S. Pat. App. Pub. No. 2007/01 17772, "Methods for slowing familial ALS disease progression," incorporated herein by reference in its entirety.
  • administration can be with one or more agents capable of promoting penetration of the subject antisensc oligonucleotide across the blood-brain barrier.
  • Injection can be made, e.g., in the entorhinal cortex or hippocampus. Delivery of neurotrophic factors by administration of an adenovirus vector to motor neurons in muscle tissue is described in, e.g., U.S. Pat. No. 6,632,427, "Adcnoviral-vcctor-mediatcd gene transfer into medullary motor neurons,” incorporated herein by reference.
  • vectors directly to the brain e.g., the striatum, the thalamus, the hippocampus, or the substantia nigra
  • Delivery of vectors directly to the brain is known in the art and described, e.g., in U.S. Pat. No. 6,756,523, "Adenovirus vectors for the transfer of foreign genes into cells of the central nervous system particularly in brain," incorporated herein by reference.
  • Administration can be rapid as by injection or made over a period of time as by slow infusion or administration of slow release formulations.
  • the subject antisense oligonucleotides can also be linked or conjugated with agents that provide desirable pharmaceutical or pharmacodynamic properties.
  • the antisense oligonucleotide can be coupled to any substance, known in the art to promote penetration or transport across the blood-brain barrier, such as an antibody to the transferrin receptor, and administered by intravenous injection.
  • the antisense compound can be linked with a viral vector, for example, that makes the antisense compound more effective and/or increases the transport of the antisense compound across the blood-brain barrier.
  • Osmotic blood brain barrier disruption can also be accomplished by, e.g., infusion of sugars including, but not limited to, meso erythritol, xylitol, D(+) galactose, D(+) lactose, D(+) xylose, dulcitol, myo-inositol, L(-) fructose, D(-) mannitol, D(+) glucose, D(+) arabinosc, D(-) arabinosc, cellobiose, D(+) maltose, D(+) raffinose, L(+) rhamnosc, D(+) melibiose, D(-) ribose, adonitol, D(+) arabitol, L(-) arabitol, D(+) fucosc, L(-) fucosc, D(-) lyxose, L(+) lyxo
  • the subject antisense compounds may be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, for example, liposomes, receptor-targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption.
  • cationic lipids may be included in the formulation to facilitate oligonucleotide uptake.
  • LIPOFECTIN available from GIBCO-BRL, Bethesda, MD.
  • Oligonucleotides with at least one 2'-0-mcthoxyethyl modification arc believed to be particularly useful for oral administration.
  • Phannaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders.
  • Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
  • Coated condoms, gloves and the like may also be useful.
  • the pharmaceutical formulations of the present invention may be prepared according to conventional techniques well known in the phannaceutical industry. Such techniques include the step of bringing into association the active ingredients with the phannaceutical carrier(s) or excipient(s). In general, the formulations arc prepared by unifonnly and intimately bringing into association the active ingredients with liquid earners or finely divided solid carriers or both, and then, if necessary, shaping the product.
  • the compositions of the present invention may be fonnulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas.
  • compositions of the present invention may also be fonnulated as suspensions in aqueous, non-aqueous or mixed media.
  • Aqueous suspensions may further contain substances that increase the viscosity of the suspension including, for example, sodium carboxymethylccllulose, sorbitol and/or dcxtran.
  • the suspension may also contain stabilizers.
  • compositions of the present invention include, but are not limited to, solutions, emulsions, foams and liposome-containing formulations.
  • the pharmaceutical compositions and formulations of the present invention may comprise one or more penetration enhancers, carriers, excipicnts or other active or inactive ingredients.
  • Emulsions are typically heterogeneous systems of one liquid dispersed in another in the fonn of droplets usually exceeding 0.1 ⁇ in diameter. Emulsions may contain additional components in addition to the dispersed phases, and the active drug that may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. icroemulsions are included as an embodiment of the present invention. Emulsions and their uses arc well known in the art and are further described in U.S. Pat. No. 6,287,860.
  • Formulations of the present invention include liposomal formulations.
  • liposome means a vesicle composed of amphophilic lipids arranged in a spherical bilayer or bilaycrs. Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior that contains the composition to be delivered. Cationic liposomes arc positively charged liposomes that arc believed to interact with negatively charged DNA molecules to fonn a stable complex. Liposomes that arc pH-sensitivc or negatively-charged arc believed to entrap DN A rather than complex with it. Both cationic and noncationic liposomes have been used to deliver DNA to cells.
  • Liposomes also include "stcrically stabilized" liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids. When incorporated into liposomes, these specialized lipids result in liposomes with enhanced circulation lifetimes relative to liposomeslacking such specialized lipids. Examples of stcrically stabilized liposomes are those in which part of the vcsicle-fonning lipid portion of the liposome comprises one or more glycolipids or is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety.
  • PEG polyethylene glycol
  • the phannaceutical formulations and compositions of the present invention may also include surfactants.
  • surfactants used in drug products, formulations and in emulsions is well known in the art. Surfactants and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein by reference.
  • the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly oligonucleotides.
  • penetration enhancers In addition to aiding the diffusion of non-lipophilic dmgs across cell membranes, penetration enhancers also enhance the pcmicability of lipophilic dmgs.
  • Penetration enhancers may be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non- chelating nonsurfactants. Penetration enhancers and their uses arc further described in U.S. Pat. No. 6,287,860, which is incorporated herein by reference.
  • formulations are routinely designed according to their intended use, i.e. route of administration.
  • Preferred formulations for topical administration include those in which the oligonucleotides of the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants.
  • a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants.
  • Preferred lipids and liposomes include neutral (e.g. diolcoyl-phosphatidyl DOPE ethanolaminc, dimyristoylphosphaudyl choline DMPC, distcarolyphosphatidyl choline) negative (e.g. dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g. dioleoyltctramcthylaminopropyl DOTAP and dioleoyl-phosphatidyl ethanolamin
  • oligonucleotides of the invention may be encapsulated within liposomes or may form complexes thereto, in particular to cationic liposomes.
  • oligonucleotides may be complexed to lipids, in particular to cationic lipids.
  • Preferred fatty acids and esters, pharmaceutically acceptable salts thereof, and their uses are further described in U.S. Pat. No. 6,287,860.
  • compositions and formulations for oral administration include powders or granules, microparticulatcs, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablcts. Thickeners, flavoring agents, diluents, cmulsificrs, dispersing aids or binders may be desirable.
  • Preferred oral formulations are those in which oligonucleotides of the invention arc administered in conjunction with one or more penetration enhancers surfactants and chelators.
  • Preferred surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof.
  • Preferred bile acids/salts and fatty acids and their uses arc further described in U.S. Pat. No. 6,287,860, which is incorporated herein by reference.
  • Also preferred arc combinations of penetration enhancers for example, fatty acids/salts in combination with bile acids/salts. A particularly preferred combination is the sodium salt of lauric acid, capric acid and UDCA.
  • Further penetration enhancers include polyoxyethylcne-9-lauryl ether, polyoxyethylenc-20-cetyl ether.
  • Oligonucleotides of the invention may be delivered orally, in granular form including sprayed dried particles, or complexed to form micro or nanoparticlcs. Oligonucleotide complcxing agents and their uses arc further described in U.S. Pat. No. 6,287,860, which is incorporated herein by reference.
  • compositions and formulations for parenteral, intrathecal or intraventricular administration may include sterile aqueous solutions that may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.
  • Certain embodiments of the invention provide pharmaceutical compositions containing one or more oligomeric compounds and one or more other chcmothcrapcutic agents that function by a non-antisense mechanism.
  • chcmothcrapcutic agents include but arc not limited to cancer chcmothcrapcutic drugs such as daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicia, esorubicia, bleomycin, mafosfamidc, ifosfamidc, cytosinc arabinosidc, bischlorocthyl- nitrosurca, busulfan, mitomycin C, actinomycin D, mithramycin, prednisone, hydroxyprogestcronc, testosterone, tamoxifen, dacarbazinc, procarbazine, hcxamcthylmclaminc, pcntamethylmel
  • chemothcrapeutic agents When used with the compounds of the invention, such chemothcrapeutic agents may be used individually (e.g., 5-FU and oligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for a period of time followed by MTX and oligonucleotide), or in combination with one or more other such chemothcrapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or 5-FU, radiotherapy and oligonucleotide).
  • chemothcrapeutic agents may be used individually (e.g., 5-FU and oligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for a period of time followed by MTX and oligonucleotide), or in combination with one or more other such chemothcrapeutic agents (e.g., 5-FU, MTX and oligonucleotide,
  • Antiinflammatory drugs including but not limited to nonsteroidal anti-inflammatory drugs and corticosteroids, and antiviral drugs, including but not limited to ribivirin, vidarabine, acyclovir and ganciclovir, may also be combined in compositions of the invention. Combinations of antisense compounds and other non-antisense drugs are also within the scope of this invention. Two. or more combined compounds may be used together or sequentially.
  • compositions of the invention may contain one or more antisense compounds, particularly oligonucleotides, targeted to a first nucleic acid and one or more additional antisense compounds targeted to a second nucleic acid target.
  • the first target may be a particular antisense sequence of RNasc H I
  • the second target may be a region from another nucleotide sequence.
  • compositions of the invention may contain two or more antisense compounds targeted to different regions of the same R ase H 1 nucleic acid target. Numerous examples of antisense compounds arc illustrated herein and others may be selected from among suitable compounds known in the art. Two or more combined compounds may be used together or sequentially.
  • compositions and their subsequent administration are believed to be within the skill of those in the art. Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of dnig accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be estimated based on EC50s found to be effective in vitro and in vivo animal models.
  • dosage is from 0.01 ⁇ g to 100 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly, or even once every 2 to 20 years. Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues. Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the oligonucleotide is administered in maintenance doses, ranging from 0.01 ⁇ g to 100 g per kg of body weight, once or more daily, to once every 20 years.
  • a patient is treated with a dosage of drug that is at least about I , at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, or at least about 100 mg kg body weight.
  • Certain injected dosages of antisense oligonucleotides are described, e.g., in U.S. Pat. No. 7,563,884, "Antisense modulation of PTP1 B expression," incorporated herein by reference in its entirety.
  • Example 1 Design of antisense oligonucleotides specific for a nucleic acid molecule antisense to an RNase HI and/or a sense strand of RNASE HI polynucleotide
  • oligonucleotide specific for or “oligonucleotide targets” refers to an oligonucleotide having a sequence (i) capable of forming a stable complex with a portion of the targeted gene, or (ii) capable of forming a stable duplex with a portion of an mR A transcript of the targeted gene.
  • Selection of appropriate oligonucleotides is facilitated by using computer programs that automatically align nucleic acid sequences and indicate regions of identity or homology. Such programs are used to compare nucleic acid sequences obtained, for example, by searching databases such as GenBank or by sequencing PCR products. Comparison of nucleic acid sequences from a range of species allows the selection of nucleic acid sequences that display an appropriate degree of identity between species. In the case of genes that have not been sequenced, Southern blots are performed to allow a determination of the degree of identity between genes in target species and other species. By performing Southern blots at varying degrees of stringency, as is well known in the art, it is possible to obtain an approximate measure of identity.
  • An antisense compound is "specifically hybridizable" when binding of the compound to the target nucleic acid interferes with the normal function of the target nucleic acid to cause a modulation of function and/or activity, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target nucleic acid sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and under conditions in which assays are performed in the case of in vitro assays
  • the hybridization properties of the oligonucleotides described herein can be determined by one or more in vitro assays as known in the art.
  • the properties of the oligonucleotides described herein can be obtained by determination of binding strength between the target natural antisense and a potential drug molecules using melting curve assay.
  • the binding strength between the target natural antisense and a potential drug molecule can be estimated using any of die established methods of measurin the strength of intermoleculai' interactions, for example, a melting curve assay.
  • Melting curve assay determines the temperature at which a rapid transition from double-stranded to single- stranded conformation occurs for the natural antisense/Molecule complex. This temperature is widely accepted as a reliable measure of the interaction strength between the two molecules.
  • a melting curve assay can be performed using a cDN A copy of the actual natural antisense RNA molecule or a synthetic DNA or RNA nucleotide corresponding to the binding site of the Molecule.
  • Multiple kits containing all necessary reagents to perform this assay are available (e.g. Applied Biosystems Inc. MeltDoctor kit). These kits include a suitable buffer solution containing one of the double strand DNA (dsDNA) binding dyes (such as ABI HRM dyes, SYBR Green, SYTO, etc.).
  • dsDNA double strand DNA
  • the properties of the dsDNA dyes are such that they emit almost no fluorescence in free form, but are highly fluorescent when bound to dsDNA.
  • the cDNA or a corresponding oligonucleotide arc mixed with Molecule in concentrations defined by die particular manufacturer's protocols.
  • the mixture is heated to 95 °C to dissociate all preformed dsDNA complexes, then slowly cooled to room temperature or other lower temperature defined by the kit manufacturer to allow the DNA molecules to anneal.
  • the newly formed complexes are then slowly heated to 95 °C with simultaneous continuous collection of data on the amount of fluorescence diat is produced by the reaction.
  • the fluorescence intensity is inversely proportional to the amounts of dsDNA present in the reaction.
  • the data can be collected using a real time PCR instrument compatible with the kit (c.g.ABl's StcpOnc Plus Real Time PCR System or lightTyper instrument, Roche Diagnostics, Lcwcs, UK).
  • Melting peaks are constructed by plotting the negative derivative of fluorescence with respect to temperature (-d(Fluorescence)/dT) on the y-axis) against temperature (x-axis) using appropriate software (for example lightTyper (Roche) or SDS Dissociation Curve, ABI). The data is analyzed to identify the temperature of the rapid transition from dsDNA complex to single strand molecules. This temperature is called Tm and is directly proportional to the strength of interaction between the two molecules. Typically, Tm will exceed 40 °C.
  • HcpG2 cells from ATCC (cat# HB-8065) were grown in growth media (MEM/EBSS (Hyclonc cat #SH30024, or Mcdiatech cat # MT- 10-O lO-CV) + 10% FBS (Mcdiatcch cat# MT35-01 1-CV)+ penicillin/streptomycin (Mediatech cat# MT30-002-CI)) at 37°C and 5% C0 2 .
  • MEM/EBSS Hyclonc cat #SH30024, or Mcdiatech cat # MT- 10-O lO-CV
  • FBS Mediatcch cat# MT35-01 1-CV
  • penicillin/streptomycin Mediatech cat# MT30-002-CI
  • the cD A from this reverse transcription reaction was used to monitor gene expression by real time PCR using ABI Taqman Gene Expression Mix (cat#4369510) and primers/probes designed by ABI (Applied Biosystcms Taqman Gene Expression Assay: Hs()0268000_m l by Applied Biosystcms Inc., Foster City CA).
  • the following PCR cycle was used: 5 ( )°C for 2 min, 95°C for 10 min, 40 cycles of (95°C for 15 seconds, 60°C for 1 min) using Mx4000 thermal cycler (Stratagene). Fold change in gene expression after treatment with antisense oligonucleotides was calculated based on the difference in 18S-normalized dCt values between treated and mock-transfcctcd samples.

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Abstract

La présente invention concerne des oligonucléotides antisens qui modulent l'expression et/ou la fonction de la RNase H1, en particulier, en ciblant les polynucléotides antisens naturels de la RNase H1. L'invention concerne également l'identification de ces oligonucléotides antisens et leur utilisation dans le traitement de maladies et de troubles associés à l'expression de la RNase H1.
PCT/US2011/022304 2010-01-25 2011-01-25 Traitement de maladies liées à la rnase h1 par l'inhibition du produit de transcription naturel antisens de la rnase h1 WO2011091390A2 (fr)

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CA2786568A CA2786568A1 (fr) 2010-01-25 2011-01-25 Traitement de maladies liees a la rnase h1 par l'inhibition du produit de transcription naturel antisens de la rnase h1
RU2012127702A RU2611192C2 (ru) 2010-01-25 2011-01-25 ЛЕЧЕНИЕ ЗАБОЛЕВАНИЙ, СВЯЗАННЫХ С РНКазой Н1, ПУТЕМ ИНГИБИРОВАНИЯ ПРИРОДНОГО АНТИСМЫСЛОВОГО ТРАНСКРИПТА К РНКазе Н1
KR1020127018234A KR101853510B1 (ko) 2010-01-25 2011-01-25 Ｒｎａｓｅ ｈ1에 대한 천연 안티센스 전사체의 억제에 의한 ｒｎａｓｅ ｈ1과 관련된 질환의 치료
JP2012550201A JP5981850B2 (ja) 2010-01-25 2011-01-25 ＲＮａｓｅＨ１に対する天然アンチセンス転写物の阻害によるＲＮａｓｅＨ１関連疾患の治療
ES11735332.6T ES2671877T3 (es) 2010-01-25 2011-01-25 Tratamiento de enfermedades relacionadas con la RNASA (H1) mediante inhibición del transcrito antisentido natural a RNASA H1
NO11735332A NO2529015T3 (fr) 2010-01-25 2011-01-25
CN2011800062572A CN102782135A (zh) 2010-01-25 2011-01-25 通过抑制rna酶h1的天然反义转录物而治疗rna酶h1相关疾病
DK11735332.6T DK2529015T3 (en) 2010-01-25 2011-01-25 TREATMENT OF RNASE H1-RELATED DISEASES BY INHIBITION OF NATURAL ANTISENSE TRANSCRIPT TO RNASE H1
EP11735332.6A EP2529015B1 (fr) 2010-01-25 2011-01-25 Traitement de maladies liées à la rnase h1 par l'inhibition du produit de transcription naturel antisens de la rnase h1
US13/575,206 US8946182B2 (en) 2010-01-25 2011-01-25 Treatment of RNASE H1 related diseases by inhibition of natural antisense transcript to RNASE H1
US14/573,326 US9745582B2 (en) 2010-01-25 2014-12-17 Treatment of RNASE H1 related diseases by inhibition of natural antisense transcript to RNASE H1
US15/684,234 US10337013B2 (en) 2010-01-25 2017-08-23 Treatment of RNASE H1 related diseases by inhibition of natural antisense transcript to RNASE H1

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US14/573,326 Division US9745582B2 (en) 2010-01-25 2014-12-17 Treatment of RNASE H1 related diseases by inhibition of natural antisense transcript to RNASE H1

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US10837014B2 (en) 2012-05-16 2020-11-17 Translate Bio Ma, Inc. Compositions and methods for modulating SMN gene family expression
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RU2012127702A (ru) 2014-03-10
EP2529015B1 (fr) 2017-11-15
ES2671877T3 (es) 2018-06-11
US9745582B2 (en) 2017-08-29
CN102782135A (zh) 2012-11-14
US20150133525A1 (en) 2015-05-14
DK2529015T3 (en) 2018-02-26
US20170355996A1 (en) 2017-12-14
CA2786568A1 (fr) 2011-07-28
JP2013517778A (ja) 2013-05-20
KR101853510B1 (ko) 2018-06-20
US10337013B2 (en) 2019-07-02
EP2529015A2 (fr) 2012-12-05
JP5981850B2 (ja) 2016-08-31
US20120295959A1 (en) 2012-11-22
US8946182B2 (en) 2015-02-03
NO2529015T3 (fr) 2018-04-14
RU2611192C2 (ru) 2017-02-21
WO2011091390A3 (fr) 2011-12-29
KR20120113755A (ko) 2012-10-15

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